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WO2018204226A1 - Compositions et procédés de prévention et de traitement de perte d'audition - Google Patents

Compositions et procédés de prévention et de traitement de perte d'audition Download PDF

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Publication number
WO2018204226A1
WO2018204226A1 PCT/US2018/030114 US2018030114W WO2018204226A1 WO 2018204226 A1 WO2018204226 A1 WO 2018204226A1 US 2018030114 W US2018030114 W US 2018030114W WO 2018204226 A1 WO2018204226 A1 WO 2018204226A1
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WIPO (PCT)
Prior art keywords
inhibitor
egfr
cas
expression
adenoviral
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PCT/US2018/030114
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English (en)
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WO2018204226A8 (fr
Inventor
Jian Zuo
Fei Zheng
Tetsuji Yamashita
Zoe KELLARD
Tal Teitz
Jaeki Min
Taosheng CHEN
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St. Jude Children's Research Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by St. Jude Children's Research Hospital filed Critical St. Jude Children's Research Hospital
Priority to US16/610,170 priority Critical patent/US11883491B2/en
Priority to EP18794717.1A priority patent/EP3618807A4/fr
Priority to CN201880029618.7A priority patent/CN111655228B/zh
Priority to JP2019560674A priority patent/JP7291399B2/ja
Publication of WO2018204226A1 publication Critical patent/WO2018204226A1/fr
Publication of WO2018204226A8 publication Critical patent/WO2018204226A8/fr
Priority to JP2022176126A priority patent/JP7515910B2/ja
Priority to JP2024102725A priority patent/JP2024153624A/ja

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    • A—HUMAN NECESSITIES
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Definitions

  • the ear is a complex organ composed of a labyrinth of structures responsible for hearing and balance. Perception of both hearing and balance lies in the ability of inner ear structures to transform mechanical stimuli to impulses recognized by the brain.
  • the sensory receptors responsible for hearing are located in the cochlea, a spiral-shaped canal filled with fluid.
  • the cochlea Within the cochlea is the organ of Corti, which is lined with columnar sensory hair cells bridging the basilar membrane and the tectorial membrane. As sound waves pass through the organ of Corti, the basilar membrane vibrates causing the hair cells to bend back and forth. The movement depolarizes the hair cell, leading to release of neurotransmitters to the auditory nerve, which carries the impulse to the brain.
  • Atonal BHLH Transcription Factor 1 (Atohl) , a lineage- specific transcription factor for sensory hair cells, directly converts non-sensory supporting cells to sensory hair cells in cochlear explant culture and in vivo (Gubbels, et al . (2008) Nature 455 ( 7212 ) : 537-41 ; Kelly, et al. (2012) J. Neurosci. 32 (19) : 6699-710; Liu, et al . (2012) J. Neurosci. 32 ( 19) : 6600-10 ; Liu, et al .
  • Atohl-mediated non-sensory supporting cell-to-sensory hair cell conversion in vivo is efficient and complete and whether such conversion bypasses the progenitor-cell state or follows normal developmental lineage paths precisely.
  • Atohl- converted sensory hair cells exhibited immature hair cell morphology and did not express several terminal differentiation markers (e.g., Slc26a5 encoding prestin and Ocm encoding oncomodulin) , and the conversion rate was low (6%-20%) (Kelly, et al . (2012) J. Neurosci. 32(19) : 6699- 710; Liu, et al . (2012) J. Neurosci. 32 ( 19 ) : 6600-10 ; Liu, et al. (2014) PLoS One 9 (2 ) : e89377 ) .
  • terminal differentiation markers e.g., Slc26a5 encoding prestin and Ocm encoding oncomodulin
  • epidermal growth factor receptor (EGFR) signaling has been shown to be required for proliferation and down-regulation of the cell cycle inhibitor p27Kipl (CDKNlb) to enable cell cycle re ⁇ entry (White, et al . (2012) Dev. Biol. 363:191-200) .
  • the invention provides a method for the treatment or prevention of hearing loss by administering to an animal in need thereof an inhibitor of epidermal growth factor receptor (EGFR) signaling.
  • the method can further include administering an expression vector harboring a nucleic acid molecule encoding an atonal-associated factor and/or other regenerative agents.
  • the method further includes administering one or more otoprotective or regenerative agents.
  • the inhibitor of EGFR signaling inhibits the expression or activity of EGFR, Ras, Raf, MEK, ERK/MAPK, JAK, STAT, PI3K, AKT, mTOR, NCK, PAK, JNK, PLC, PKC or a cell cycle-associated protein kinase inhibitor (e.g., Her-2, Aurora Kinase, B-Raf or PDGFR) .
  • the inhibitor is an inhibitory RNA, antibody or small organic molecule.
  • Pharmaceutical compositions and kits containing an expression vector harboring a nucleic acid molecule encoding an atonal- associated factor in combination with an inhibitor of epidermal growth factor receptor (EGFR) signaling and optionally one or more regenerative agents are also provided .
  • Figures 1A-1D show that pharmacological inhibition of the EGFR signaling increased Atohl-induced hairy cell
  • HC HC conversion in neonatal mouse cochlear explants.
  • Representative images show the immunostaining of cochlear explants transfected with Atohl-IRES-GFP and treated with vehicle ( Figures 1A and 1C) or ⁇ AG1478 ( Figures IB and ID) .
  • Expression of hair cell marker Myo6 ( ⁇ ) and Atohl- transfected cells (GFP, i) are shown.
  • Figures 1C and ID represent high magnifications of the square areas in Figures 1A and IB, respectively. Scale bar: 100 pm in Figures 1 ⁇ and IB, 20 pm in Figures 1C and ID.
  • Atohl is transfected (O/E) in all conditions.
  • FIG. 3 shows that the EGFR inhibitor MUBRITINIB (whose structure is shown) protects against cisplatin- induced hair cell loss in mouse cochlear explants with IC 50 of 2.5 nM and LD 50 of >500 nM (Therapeutic Index of >200). Number of explants: 1-4 at each dose; FVB cochlear explants with 150 pM cisplatin treatment and middle turns were analyzed; curve fitting with R 2 of 0.86. Note that IC 50 values of MUBRITINIB were consistent in all assays demonstrating its specificity and potency.
  • FIG. 4 shows that the EGFR inhibitor Pelitinib (whose structure is shown) protects against cisplatin- induced hair cell loss.
  • Pelitinib is an irreversible inhibitor of EGFR that exhibits protective effects against cisplatin-induced Caspase-3/7 activity in HEI-OCl cells with IC 50 of 0.6 pM (cisplatin-Glo 3/7) and LD 50 of >40 pM (CELLTITER-GLO) .
  • this inventive provides compositions and methods for the prevention of hearing loss using an inhibitor of EGFR and/or treatment of hearing loss using an inhibitor of EGFR in combination with Atonal-associated factor gene therapy.
  • the inventive methods prophylactically or therapeutically treat an animal, preferably a mammal (e.g., a human), for at least one disorder associated with loss or damage of sensory hair cells, e.g., disorders of the ear associated with damage of sensory hair cells (such as hearing loss or balance disorders) .
  • the inventive methods also are useful in maintaining a level of sensory perception, i.e., controlling the loss of perception of environmental stimuli caused by, for instance, the aging process or ototoxic agents .
  • EGFR Epidermal growth factor receptor
  • ErbB-1 HER1 in humans
  • EGFR is a cell-surface receptor activated by binding of its specific ligands, including epidermal growth factor (EGF) , transforming growth factor a (TGF ) , HB-EGF, amphiregulin, betacellulin, epigen, and epiregulin.
  • EGF epidermal growth factor
  • TGF transforming growth factor a
  • HB-EGF transforming growth factor a
  • amphiregulin betacellulin
  • betacellulin epigen
  • epiregulin epiregulin
  • EGFR is a member of the ErbB family of receptors, a subfamily of four closely related receptor tyrosine kinases: EGFR, HER2/c-neu (ErbB-2), Her3 (ErbB-3) and Her4 (ErbB-4) .
  • EGFR Upon activation by its growth factor ligands, EGFR undergoes a transition from an inactive monomeric form to an active homodimer. In addition to forming homodimers after ligand binding, EGFR can pair with another member of the ErbB receptor family, such as ErbB2/Her2/neu, to create an activated heterodimer. EGFR dimerization stimulates its intrinsic intracellular protein-tyrosine kinase activity. As a result, autophosphorylation of several tyrosine (Y) residues in the C-terminal domain of EGFR occurs. These include Y992, Y1045, Y1068, Y1148 and Y1173.
  • Y tyrosine
  • This autophosphorylation elicits downstream activation, signaling and/or expression of Ras/Raf/MEK/ERK/MAPK, JAK/STAT, Pl3K/AKT/mTOR, NCK-PAK- JNK, PLC-DAG-PKC and/or a number of cell cycle-associated protein kinase proteins/pathways.
  • These signaling events initiate several signal transduction cascades leading to DNA synthesis and cell migration, adhesion, and proliferation.
  • EGFR signaling or “an EGFR signaling pathway” refers herein to signaling by EGFR itself, as well as the Ras/Raf/MEK/ERK/MAPK, JAK/STAT, PI3K/AKT/mTOR, NCK-PAK-JNK, PLC-DAG-PKC, cell cycle- associated protein kinase pathways/proteins downstream thereof .
  • Ras/Raf/MEK/ERK/MAPK Pathway The Ras/Raf/MEK/ERK/MAPK pathway (also known as the MAPK/ERK pathway) is well-known in the art and plays a central role in regulating mammalian cell growth by relaying extracellular signals from ligand-bound cell surface tyrosine kinase receptors such as EGFR. Activation of the MAPK/ERK
  • Ras mitogen-activated protein kinase/extracellular signal- regulated kinase pathway is via a cascade of phosphorylation events that begins with activation of Ras, e.g., HRas (GENBANK Accession No. NP_001123914 , NP_001304983, or NP_789765), KRas (GENBANK Accession No. NP_004976 or NP_203524) or NRas (GENBANK Accession No. NP_002515) .
  • Activation of Ras leads to the recruitment and activation of Raf, e.g., c-Raf or Raf-1 (GENBANK Accession No.
  • NP_002871 A-Raf (GENBANK Accession No. NP_001243125, NP_001645 or NP_001243126) or B-Ra f (GENBANK Accession No. NP_004324) .
  • Activated Raf then phosphorylates and activates MEK1/2 (i.e., MAPK/ERK Kinase-1 and -2; GENBANK Accession Nos. NP_002746 and NP_109587, respectively), which then phosphorylates and activates ERK1/2 (i.e., MAPK3/MAPKl ; UniProt Accession Nos. P28482 and P27361, respectively).
  • This chain of proteins communicates signals from cell-surface receptors to the DNA.
  • ERK generates extensive changes in gene expression mediated by transcription factors that control cell cycle progression, differentiation, protein synthesis, metabolism, cell survival, cell migration, and invasion and senescence.
  • JAK/STAT Pathway The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway stimulates cell proliferation, differentiation, cell migration and apoptosis.
  • JAK/STAT signaling is composed of a few principal components.
  • the JAK family includes four members: JAKl (GENBANK Accession No. NP_001307852 ) , JAK2 (GENBANK Accession No. NP_001309123 or NP_001309127 ) , JAK3 (GENBANK Accession No. NP_000206) and Tyk2 (GENBANK Accession No. NP_003322) .
  • STATs are latent transcription factors that reside in the cytoplasm until activated.
  • the mammalian STATs i.e., STAT1, GENBANK Accession No. NP_009330 or NP_644671; STAT2, GENBANK Accession No. NP_005410 or NP_938146; STAT3 , GENBANK Accession No. NP_003141, NP_644805, or NP_998827; STAT4, GENBANK Accession No.
  • PI3K/AKT/mTOR Pathway The PI3K/AKT/mTOR pathway is an intracellular signaling pathway important in regulating the cell cycle. Ligand-bound activation of EGFR leads to the activation of PI3K (phosphatidylinositol-4 , 5- bisphosphate 3-kinase, e.g., Class 1 enzymes such as PIK3CA, PIK3CB, PIK3CG, PIK3CD, PIK3R1, PIK3R2, PIK3R3, PIK3R4, PIK3R5 and PIK3R6; Class 2 enzymes such as PIK3C2A, PIK3C2B and PIK3C2G; and Class 3 enzyme PIK3C3) .
  • PI3K phosphatidylinositol-4 , 5- bisphosphate 3-kinase
  • Class 1 enzymes such as PIK3CA, PIK3CB, PIK3
  • PI3K subsequently phosphorylates Akt ⁇ i.e., Protein Kinase B or PKB including AKT1 , UniProt Accession No. P31749; AK 2 , UniProt Accession No. P31751; and AKT3, UniProt Accession No. Q9Y243) .
  • PIK3 subsequently activates mTOR complexes, mTORCl and mTORC2, which are each involved in cell growth.
  • mTORCl which is composed of mTOR, Raptor, G L (mammalian lethal with SEC13 protein 8) and domain-containing mTOR- interacting protein (DEPTOR) , unifies multiple signals that indicate the availability of growth factors, nutrients and energy in order to promote cellular growth and catabolic processes during stress.
  • Active mTORCl exerts numerous downstream biological effects, including the translation of mRNA by phosphorylating downstream targets, such as 4E-BP1 and p70 S6 kinase, the suppression of autophagy through Atgl3 and ULKl, ribosome biogenesis, and activation of transcription that leads to increased mitochondrial activity or adipogenesis .
  • mTORC2 which is composed of mTOR, Rictor, ⁇ -, Sinl, PRR5/Protor-l and DEPTOR, promotes cell survival through the activation of Akt.
  • mTORC2 regulates cytoskeletal dynamics, ion transport and growth by activating PKC and phosphorylating SGKl.
  • Nek non-catalytic region of tyrosine kinase adaptor protein 1; GENBANK Accession No. NP_001177725 or NP_001278928 ) is known to bind to activated EGFR through its SH2 domain. Nek associates with PAKl (p21/CDC42/Racl-Activated Kinase-1; GENBANK Accession No. NP_001122092 or NP_002567) through the first N-terminal polyproline domain of PAKl and an SH3 domain of Nek.
  • PAKl p21/CDC42/Racl-Activated Kinase-1
  • Nek activates PAK, which subsequently activates JNKs (c-Jun Kinases) via MEKKl (MAP/ERK Kinase Kinase-1; GENBANK Accession No. NP_005912) and KK4/7 (MAP Kinase Kinase-4/7; GENBANK Accession No. NP_1268364, NP_003001, NP_001284484 , or NP_001284485) .
  • Activated JNKs enter the nucleus and cause phosphorylation of transcription factors such as c- Fos and c-Jun.
  • PLC-DAG-PKC Pathway Phospholipase C (PLC) ties EGFR activation to the generation of secondary messengers and calcium metabolism. EGFR recruits and phosphorylates PLC- ⁇ (GENBANK Accession No. NP_002651 or NP_877963), which then generates diacylglycerol (DAG) and inositol-1 , 4 , 5- trisphosphate (IP3) from Ptdlns ( 4 , 5 ) P2. DAG activates many isoforms of Protein kinase C (PKC), including conventional isoforms , ⁇ , and ⁇ , as well as PKC- ⁇ and PKC- ⁇ .
  • PLC-DAG-PKC Pathway Phospholipase C (PLC) ties EGFR activation to the generation of secondary messengers and calcium metabolism. EGFR recruits and phosphorylates PLC- ⁇ (GENBANK Accession No. NP_002651 or NP_877963)
  • PKC- , PKC- ⁇ , PKC- ⁇ , and PKC- ⁇ phosphorylate and activate c-Raf-1, thereby amplifying HRas/MEKl and MEK2/ERK1/2 kinase cascades.
  • PKC- ⁇ activates Nuclear factor NF-kappa-B inhibitor kinase beta (IKK-beta) resulting in activation of the Nuclear factor NF-kappa-B (NF-kB) .
  • Cell Cycle-Associated Protein Kinases Protein kinases downstream or interacting with EGFR play a central role in the regulation of the eukaryotic cell cycle. More specifically, these protein kinases are involved in signal transduction, chromosome condensation, centrosome maturation, spindle assembly, spindle orientation, meiotic maturation, and cytokinesis. Accordingly, a "cell-cycle associated protein kinase” refers to a kinase downstream of, or interacting with, EGFR that regulates one or more of cell cycle progression, cell division, cell proliferation, and cell cycle machinery.
  • the cell cycle-associated protein kinase of this invention is Her2/neu, Aurora kinase, B-raf (as discussed herein) or platelet-derived growth factor receptor (PDGFR),
  • Her2/neu Kinase Her2/neu is a 185-kDa transmembrane protein (GENBANK Accession No. NP_001005862 , NP_001276865, NP_001276866, NP_001276867 , or NP_004439) encoded by the erbB2 oncogene located on chromosome 17q21-22. Normal expression of Her2/neu at the cell surface is essential for regulating cell growth and epithelial cell survival. While a natural ligand of Her2/neu has not been identified, Her2/neu is known to be a preferred dimerization partner forming potent heterodimers with EGFR and Her3 (Lenferink, et al. (1998) EMBO J. 17:3385-97) .
  • Aurora Kinase The Aurora kinases are a family of highly conserved serine/threonine kinases that are important for faithful transition through mitosis
  • Aurora A maps to chromosome region 20ql3.2, a region that has been found amplified in different human cancers. Aurora A (GENBANK Accession No.
  • NP_001310232 , NP_001310233, NP_001310234, NP_003591 or NP_940835 plays an important role in centrosome maturation, spindle assembly, meiotic maturation, and metaphase I spindle orientation (Carmena & Earnshaw (2003) Nat. Rev. Mol. Cell Biol. 4:842-54).
  • Aurora A function is regulated by degradation, phosphorylation, and dephosphorylation, with its kinase activity dependent upon phosphorylation of threonine 288 (Thr288) in the activation loop.
  • Selective inhibition of Aurora A results in inhibition of autophosphorylation of Aurora A at Thr288, monopolar spindles, and G2-M arrest (Girdler, et al. (2006) J.
  • the Aurora B (GENBANK Accession No. NP_001243763, NP_001271455, NP_001300879, NP_001300880, or NP_001300881) gene maps to chromosome region 17pl3.1 and this kinase forms part of the chromosomal passenger complex (CPC) with three non- enzymatic subunits: inner centromere protein (INCENP), Survivin, and Borealin (Vader, et al . (2006) J. Cell Biol. 173:833-7).
  • CPC chromosomal passenger complex
  • the highly dynamic CPC is critical for chromosome condensation, chromosome orientation on the mitotic spindle, through correcting chromosome-microtubule attachment errors, and the spindle-assembly checkpoint (SAC), as well as the final stages of cytokinesis (Sampath, et al. (2004) Cell 118:187-20; Terada, et al . (1998) EMBO J. 17:667-76; Méa, et al . (2012) Nat. Rev. Mol . Cell Biol. 13:789-803; Tanenbaum, et al . (2011) Curr. Biol. 21:1356-6).
  • Aurora C GenBANK Accession No.
  • NP_001015878 , NP_001015879, or NP_003151 expression has been reported in testis, thyroid, and placenta and in meiotically dividing gametes (Ulisse, et al . (2006) Int. J. Cancer 119:275-82; Bernard, et al . (1998) Genomics 53:406-9; Kimura, et al . (1999) J. Biol. Chem. 274:7334-40; Yang, et al . (2010) Mol. Biol. Cell 21:2371-83). Further, nuclear EGFR, associated with STAT5, has been shown to bind and increase Aurora-A gene expression (Hung, et al . (2008) Nucl. Acids Res. 36 (13) : 4337-51) . Overexpression of Aurora C has been suggested to induce abnormal cell division resulting in centrosome amplification and multinucleation in cells.
  • PDGFR Kinase PDGFR Kinase. PDGFR is involved in the control of cell proliferation, differentiation and survival in various tissues of vertebrates. Activated PDGFR phosphorylates itself and other proteins, and thereby engages intracellular signaling pathways that trigger cellular responses such as migration and proliferation. PDGFRa
  • Inhibitors of EGFR Signaling is intended to refer to any molecule that reduces, blocks or decreases the expression or activity of an EGFR protein or a protein that interacts with or is in a downstream pathway of EGFR, e.g., a Ras, Raf, MEK, ERK/MAPK, JAK, STAT, PI3K, A T, mTOR (including a protein of an mTOR complex) , NCK, PAK, JNK, PLC, PKC or cell cycle- associated protein kinase (e.g., Her-2, Aurora kinase, B- Raf, or PDGFR) .
  • An inhibitor of EGFR signaling also includes an inhibitor that blocks the expression or activity of an EGFR ligand such as EGF, TGF- , HB-EGF, AR, BTC, EPR, or epigen.
  • an EGFR ligand such as EGF, TGF- , HB-EGF, AR, BTC, EPR, or epigen.
  • the inhibitor of EGFR signaling inhibits or reduces the expression or activity of EGFR, PLC, STAT3, JAK2 , PI3K, MEK, Her-2, Aurora kinase, B-Raf, or PDGFR.
  • An inhibitor of this invention can selectively decrease or block the expression of an EGFR signaling protein (i.e., transcription or translation of the protein) , decrease or block the activity of an EGFR signaling protein ⁇ i.e., binding to ligands, tyrosine kinase activity, phosphorylation, protein-protein interactions, and/or downstream signaling), decreas ⁇ or block the biological effect (s) of an EGFR signaling protein, and/or modify half-life or subcellular localization (membrane versus cytoplasmic or nuclear localization, internalization, and recycling) of an EGFR signaling protein.
  • an EGFR signaling protein i.e., transcription or translation of the protein
  • an EGFR signaling protein i.e., transcription or translation of the protein
  • an EGFR signaling protein i.e., transcription or translation of the protein
  • an EGFR signaling protein i.e., binding to ligands, tyrosine kinase activity,
  • an inhibitor of EGFR signaling is an active agent that selectively decreases or blocks one or more of the following: transcription or translation, ligand binding, phosphorylation, multimerization, tyrosine kinase activity, internalization, and/or translocation into the nucleus.
  • EGFR signaling is completely blocked, or is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 97%, at least 98%, at least 98,5%, at least 99%, at least 99.25%, at least 99.5%, or at least 99.75% by the inhibitor of EGFR signaling inhibitor as compared to normal physiologic levels.
  • the inhibitor of EGFR signaling of this invention typically has a half maximal (50%) inhibitory concentration (IC 50 ) in the range of 1 pM to 100 ⁇ .
  • IC 50 half maximal inhibitory concentration
  • the inhibitor of EGFR signaling has an IC 50 value of less than 10 ⁇ , less than 5 ⁇ , less than 1 ⁇ , or less than 100 nM.
  • the inhibitor of EGFR signaling is specific/selective for one or more of the EGFR signaling proteins of interest and fails to inhibit, or inhibits to a substantially lesser degree other non-EGFR pathway proteins. In this respect, it is preferable that the inhibitor of EGFR signaling is a selective inhibitor of EGFR signaling.
  • selectivity is for one, two, three or four EGFR signaling proteins and fails to inhibit, or inhibits to a substantially lesser degree other non-EGFR pathway proteins.
  • an inhibitor can be a dual EGFR and ERBB2 inhibitor, both of which are EGFR signaling proteins.
  • Methods for assessing the selectively of inhibitors are known in the art and can be based upon any conventional assay including, but not limited to the determination of the IC 50 , the binding affinity of the inhibitor (i.e., Ki) , and/or the half maximal effective concentration (EC 50 ) of the inhibitor for EGFR signaling protein of interest as compared to another protein (comparative protein) .
  • a selective inhibitor of EGFR signaling is an inhibitor that has an IC 50 value for an EGFR signaling protein of interest that is at least twice or, more desirably, at least three, four, five, or six times lower than the corresponding IC 50 value for a comparative protein.
  • a selective inhibitor of EGFR signaling has an IC 50 value for an EGFR signaling protein which is at least one order of magnitude or at least two orders of magnitude lower than the IC 50 value for a comparative protein.
  • An inhibitor of this invention can be a nucleic acid-based inhibitor such as an inhibitory RNA molecule
  • antisense molecule e.g., antisense molecule, a ribozyme, siRNA, shRNA, miRNA, etc.
  • gene expression e.g., polyadenylation
  • level of expression of another gene within the cell i.e., where gene expression is broadly considered to include all steps from initiation of transcription through production of a process protein
  • gene expression is broadly considered to include all steps from initiation of transcription through production of a process protein
  • an antibody including fragments or mimetics
  • a peptide a small organic molecule; or a combination thereof .
  • siRNA refers to double stranded RNA or RNA and DMA species that are active to reduce expression of targeted gene. These molecules are known variously as “small interfering RNA,” “short interfering RNA” or “silencing RNA.” siRNA strands are usually 20-25 nucleotides long, although larger precursor molecules which are subject to cleavage in vivo to form the active species are within the scope of the term as used herein.
  • miRNA molecule is a small RNA molecule, typically about 20 to 25 nucleotides, encoded by the genome of an animal or produced synthetically with a seguence which corresponds to one encoded by the genome of the animal.
  • miRNA molecules may be single-stranded or double-stranded.
  • nucleic acid molecules encoding such an inhibitor can be carried by the same nucleic acid molecule that encodes the atonal-associated factor or can be a separate nucleic acid molecule present on the same expression vector or part of a different expression vector.
  • Inhibitory RNA molecules can be readily prepared based upon the nucleic acid seguences disclosed herein.
  • inhibitory RNA molecules such as siRNAs can be obtained from commercial sources such as Dharmacon (see, e.g., 0N- TARGET plus siRNA SMART pools), Invitrogen or Zyagen.
  • a decrease in the expression of a protein can be measured using conventional techniques such as dot blot, northern blot, ELISA or western blot analysis.
  • EGFR inhibitors that selectively decrease or block the expression of EGFR itself include, but are not limited to, EGFR antisense, siRNA and miRNA molecules.
  • EGFR antisense and siRNA that reduce the expression of EGFR are disclosed, e.g., in US 2011/0046067 and Kang, et al. (2006) Cancer Gene Ther. 13(5): 530-8.
  • miRNA that reduce the expression of EGFR are disclosed, e.g., in US 8,673,872, incorporated herein by reference in its entirety.
  • An EGFR inhibitor can also be an antibody, antibody fragment, or antibody mimetic that specifically binds EGFR and antagonizes the activity thereof by, e.g., blocking ligand binding, activation, phosphorylation or protein- protein interactions.
  • Cetuximab (IgGl) and Panitumumab (IgG2) are examples of monoclonal antibody inhibitors of EGFR.
  • Other antagonistic monoclonal antibodies include Zalutumumab, Nimotuzumab, Matuzumutab, ICR62, and mAb806.
  • Peptide inhibitors of EGFR that regulate EGFR multimerization and activation are also of use in this invention.
  • Exemplary peptide inhibitors of EGFR can be based upon the following j uxtamembrane sequence from EGFR: LLLWALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPS (SEQ ID NO : 2 ) , and may optionally include a cell penetrating component such as a protein transduction domain (PTD) to facilitate delivery into the cell.
  • PTD protein transduction domain
  • the EGFR inhibitor can be small molecule that inhibits the tyrosine kinase activity of EGFR. Without kinase activity, EGFR is unable to activate itself, which is a prerequisite for binding of downstream adaptor proteins .
  • small molecule inhibitors of EGFR include, but are not limited to, erlotinib (CAS 183321-74-6), gefitinib (CAS 184475-35-2), lapatinib (CAS 231277-92-2, dual EGFR and ERBB2 inhibitor), neratinib (CAS 698387-09-6), canertinib (CAS 267243-28-7), vandetanib (CAS 443913-73-3), afatinib (CAS 439081-18-2), AG 1478 (CAS 153436-53-4), TAK-285 (CAS 871026-44-7, dual HER2 and EGFR inhibitor) , ARRY334543 (CAS 845272-21-1, dual EGFR phosphorylation inhibitor), Dacomitinib (CAS 1110813-31-4, EGFR and ERBB2 inhibitor), AZD3759 (CAS 1626387-80-1), NT113 (CAS 1398833-56-1, pan-ERBB inhibitor), OSI-420 (Desmethyl Err
  • Ras/Raf/MEK/ERK/MAPK Inhibitors include, but are not limited to, antisense, siRNA and miRNA molecules.
  • antisense inhibition of MEKl is disclosed in US 6,096,543, incorporated herein by reference in its entirety .
  • Ras inhibitors include, but are not limited to, R115777 (CAS 192185-72-1), BMS-214662 (CAS 195987-41-8), SCH66336 (CAS 193275-84-2), FTI-277 (CAS 1217447-06-7), manumycin A (CAS 52665-74-4), FTI-276 (CAS 170006-72-1), RasCAAX (a peptidomimetic) , L-744,832 (CAS 1177806-11-9), and derivatives and combinations thereof.
  • Raf inhibitors of use in this invention include, but are not limited to, Bay43-9006 (sorafenib, CAS 284461-73-0, selective inhibitor fox B-Raf and C-Raf) , vemurafenib (CAS 918504-65-1, B-Raf inhibitor), dabrafenib (CAS 1195764-45- 7, B-Raf inhibitor; see also US 7, 994, 185 and US 8,415,345), LY3009120 (CAS 1454682-72-4, pan-Raf inhibitor), GW 5074 (CAS 220904-83-6, C-Raf-1 inhibitor), ZM 336372 (CAS 208260-29-1, Raf-1 inhibitor), 2- bromoaldisine (CAS 96562-96-8, RAF/MEK-l/MAPK pathway inhibitor), L-779,450 (CAS 303727-31-3), AZ628 (CAS 878739- 06-1, Raf-1 inhibitor), RAF265 (CAS 92
  • MEK inhibitors include, but are not limited to, SL- 327 (CAS 305350-87-2, inhibitor of MEKl and MEK2), PD 184,352 (CAS 212631-79-3), 2-bromoaldisine (CAS 96562-96-8, Raf/MEK-l/MAPK pathway inhibitor), PD 198306 (CAS 212631- 61-3, non-ATP-competitive inhibitor of MEKl/2), PD 0325901 (CAS 391210-10-9, inhibitor of MEK and suppressor of ERK phosphorylation), MEK inhibitor II (CAS 623163-52-0), PD 184161 (CAS 212631-67-9, selective inhibitor for MEKl and MEK2), U-0126 (CAS 109511-58-2, inhibitor for MEKl/2), PD 98059 (CAS 167869-21-8, selective inhibitor for MEKl), AS703026 (CAS 1236699-92-5, inhibitor for MEKl/2), BAY 869766 (CAS 923032-37-5, non
  • ERK inhibitors include, for example, SCH772984 (CAS 942183-800-4, ERKl/2 inhibitor), DEL-22379 (CAS 181223-80- 3, ERK dimerization inhibitor), VX-lle (CAS 896720-20-0, ERK2 inhibitor), Pluripotin (SCI, CAS 839707-37-8, dual ERK1 and RasGAP inhibitor) , Ulixertinib (BVD-523, VRT752271, CAS 869886-67-9, ERK1/ERK2 inhibitor), FR 180204 (CAS 865362-74-9, ATP-competitive ERK inhibitor), GDC-0994 (CAS 1453848-26-4, ERKl/2 inhibitor), KO-947 (Kura Oncology, ERKl/2 inhibitor) , and derivatives and combinations thereof. See also JP 2005-330265 for additional ERK inhibitors .
  • JAK/STAT Inhibitors that selectively decrease or block the expression of JAK or STAT include, but are not limited to, antisense, siRNA and miRNA molecules.
  • antisense inhibition of STAT-2, STAT-3, STAT-4, STAT-5 and STAT-6 is disclosed in US 2004/0101853, US 6,159,694, US 6,479,465, US 8,722,873 and WO 1998/040478, respectively.
  • siRNA for reducing the expression of STAT-1 and STAT-2 are disclosed in US 9,198,911.
  • STAT-3 siRNA are described in US 2010/0298409
  • STAT-5 siRNA are described in WO 2009/039199
  • STAT-6 siRNA are described in US 7,566,700.
  • Non-limiting examples of STAT inhibitors include, but are not limited to, WP-1034 (CAS 857064-42-7, Jak-Stat inhibitor), fludarabine (CAS 21679-14-1, STAT1 inhibitor), S3I-201 (CAS 501919-59-1, inhibitor of S AT3 DNA-binding activity), Stattic (CAS 19983-44-9, STAT3 inhibitor), APTSTAT3-9R ( STAT-binding peptide), STA-21 (CAS 28882-53-3, STAT3 inhibitor), SH-4-54 (CAS 1456632-40-8), Napabucasin (CAS 83280-65-3, STAT3 inhibitor), Cryptotanshinone (CAS 35825-57-1, STAT3 inhibitor), niclosamide (CAS 50-65-7, STAT3 inhibitor), NSC 74859 (CAS 501919-59-1, STAT3 inhibitor), NSC 74859 (CAS 501919-59-1, STAT3 inhibitor),
  • Jakl/Jak2 inhibitors include, but are not limited to, AG-490 (CAS 133550-30-8), CYT387 (CAS 1056634-68-4), SB1518 (Pacritinib, CAS 937272-79-2), LY3009104 (INCB28050, Baricitinib, CAS 1187594-09-7), TG101348 (CAS 936091-26-8), BMS-911543 (CAS 1271022-90-2), AZD1480 (CAS 935666-88-9), Ruxolitinib (INCB018424, CAS 941678-49-5), CEP-701 (CAS 111358-88-4), TG101348 (Fedratinib, CAS 936091-26-8), SD 1008 (CAS 960201-81-4, JAK2/STAT3 inhibitor), WP-1066 (CAS 857064-38-1, JAK2/STAT3 inhibitor), and derivatives and combinations thereof.
  • JAK3 inhibitors include, but are not limited to, Janex 1 ( HI-P131, CAS 202475-60-3), PF-956980 (CAS 1262832-74-5), WHI-P154 (CAS 211555-04-3), VX-509 (Decernotinib, CAS 944842-54-0), JAK3 Inhibitor IV (ZM- 39923, CAS 1021868-92-7), tofacitinib (CP-690550, CAS 540737-29-9), and derivatives and combinations thereof.
  • PI3K/AKT/mTOR Inhibitors that selectively decrease or block the expression of PI3K, AKT or mTOR include, but are not limited to, antisense, siRNA and miRNA molecules.
  • siRNA inhibition of PI3K, AKT and mTOR is disclosed in, e.g., US 2005/0272682, US 2008/0161547, and US 9,012,622 respectively .
  • Small molecules of use in inhibiting PI3K include, but are not limited to, SF1101 (LY 294002, CAS 154447-36- 6), BKM120 (CAS 944396-07-0), BYL719 (CAS 1217486-61-7), XL-147 (CAS 956958-53-5), ZSTK-474 (CAS 475110-96-4), PX- 866 (CAS 502632-66-8), PI-103 (CAS 371935-74-9), and derivatives and combinations thereof.
  • Exemplary AKT inhibitors include, e.g., AZD5363 (CAS 1143532-39-1), GDC-0068 (CAS 1001264-89-6, ATP-competitive pan-Akt inhibitor), MK-2206 (CAS 1032350-13-2), Perifosine (CAS 157716-52-4), PBI-05204 (Oleandrin, CAS 465-16-7), GSK2141795 (CAS 1047634-65-0), and SR13668 (CAS 637774-61- 9), and derivatives and combinations thereof.
  • Additional AKT inhibitors are described in US 2010/0009397, US 2007/0185152, US 6,960,584, US 7,098,208, US 7,223,738, US 7,304,063, US 7,378,403, US 7,396,832, US 7,399,764, US 7,414,055, US 7,544,677, US 7,576,209, US 7,579,355, US 7,589,068, US 7,638,530, US 7,655,649, US 7,705,014, US 7,750,151, US 7,943,732, US 8,003,643, US 8,003,651, US 8,008,317, US 8,168,652, US 8,263,357, US 8,273,782, and US 8, 324, 221.
  • Exemplary dual mTOR/PI3K inhibitors include, e.g., SF1126 (CAS 936487-67-1), BEZ235 (CAS 915019-65-7), BGT-226 (CAS 1245537-68-1), PF-04691502 (CAS 1013101-36-4), GNE-477 (CAS 1032754-81-6), XL765 (CAS 1349796-36-6), GDC-0941 (CAS 957054-30-7), GDC-0980 (CAS 1032754-93-0), PF-05212384 (CAS 1197160-78-3), and derivatives and combinations thereof.
  • SF1126 CAS 936487-67-1
  • BEZ235 CAS 915019-65-7
  • BGT-226 CAS 1245537-68-1
  • PF-04691502 CAS 1013101-36-4
  • GNE-477 CAS 1032754-81-6
  • XL765 CAS 1349796-36-6
  • GDC-0941 CAS 957054-30-7
  • Inhibition of mTOR can be achieved using one or more of the following inhibitors, e.g., OSI-027 (CAS 936890-98-
  • INK-128 (CAS 1224844-38-5), AZD-8055 (CAS 1009298-09-
  • AZD-2014 (CAS 1009298-59-2), Palomid 529 (CAS 914913- 88-5), Pp-242 (CAS 1092351-67-1), GSK2126458 (CAS 1086062- 66-9), PF-04691502 (CAS 1013101-36-4), wortmannin (CAS 19545-26-7), Ku-0063794 (CAS 938440-64-3), WAY-600 (CAS 1062159-35-6), WYE-687 (CAS 1062161-90-3), WYE-354 (CAS 1062169-56-5), rapamycin (CAS 53123-88-9), and derivatives and combinations thereof.
  • Rapamycin derivatives are further described in, e.g., US 5,258,389, US 5,100,883, US 5,118,678, US 5,151,413, US 5,256,790, US 5,120,842, US 2011/0178070, WO 1994/09010, WO 1992/05179, WO 1993/11130, WO 1994/02136, WO 1994/02485, WO 1994/02136, WO 1995/16691, WO 1996/41807, WO 1996/41807, WO 1998/02441, WO 2001/14387, and WO 1995/14023. See also US 2016/0244424 for additional PI3K/AKT/mTOR inhibitors.
  • NCK-PAK-JNK Inhibitors that selectively decrease or block the expression of NCK, PAK or JNK include, but are not limited to, antisense, siRNA and miRNA molecules.
  • siRNA inhibition of Pakl is disclosed in, e.g., WO 2013/135745.
  • siRNA inhibition of JNK1, JNK2 and JNK3 is disclosed in, e.g., US 2015/036118 .
  • Inhibitors of PAK kinases include, but are not limited to, 2-aminopyrido [2 , 3- d] pyrimidin-7 ( 8H) -ones such as those disclosed in WO 2009/086204, WO 2010/071846, WO 2011/044535, WO 2011/156646, WO 2011/156786, WO 2011/156640, WO 2011/156780, WO 2011/156775, and WO 2011/044264; 1H- thieno[3,2-c] pyrazoles , 3-amino-tet ahydropyrrole [3,4- c]pyrazoles and N4- ( lH-pyrazol-3-yl ) pyrimidine-2 , -diamines as disclosed in WO 2004/007504, WO 2007/023382, WO 2007/072153, and WO 2006/072831; N2-bicyclic indolyl, indazolyl and benz
  • JNK1, JNK2 and/or JNK3 inhibitors include, but are not limited to, JNK Inhibitor V
  • the inhibitor is not leflunomide.
  • PLC-DAG-PKC Inhibitors that selectively decrease or block the expression of PLC or PKC include, but are not limited to, antisense, siRNA and miRNA molecules.
  • siRNA inhibition of PLC is disclosed in US 9,546,367, the siRNA molecules of which are incorporated herein by reference.
  • Anti-PLCy antibodies are also known in the art for use in modulating the binding and/or catalytic activity of a PLCy. Examples of anti-PLCy antibodies are described in, for example, Lee, et al. (2002) Mol. Vis. 8:17-25 and Buckley, et al . (2004) J. Biol. Chem. 279:41807-14.
  • Examples of small molecule inhibitors of PLC include, but are not limited to, D609 (CAS 83373-60-8), edelfosine (ET-18-OCH3, CAS 77286-66-9, dual PLC/PKC inhibitor), manoalide (CAS 75088-80-1), NCDC (CAS 10556-88- 4), U-73122 (CAS 112648-68-7), and derivatives and combinations thereof.
  • Her2/neu Inhibitors that selectively decrease or block the expression of Her2/neu include, but are not limited to, antisense, siRNA and miRNA molecules.
  • siRNA inhibition of Her2/neu is disclosed in, e.g., Faltus, et al . (2004) Neoplasia 6(6) :786-95; Choudhury, et al . (2004) Int. J. Cancer 108 : 71-77.
  • Anti-Her2 /neu antibodies are also known in the art for use in modulating the activity of Her2/neu.
  • anti-Her2/neu antibodies include, but are not limited to, trastuzumab (HERCEPTIN, CAS 180288-69-1) and pertuzumab
  • Examples of small molecule inhibitors of Her2/neu include, but are not limited to, Lapatinib (TYKERP, CAS 231277-92-2, dual EGFR/Her2 inhibitor), Afatinib (GIOTRIF, CAS 439081-18-2, irreversible pan inhibitor), AZD8931 (CAS 848942-61-0, EGFR/Her2/ErbB3 inhibitor), AST-1306 (CAS 897383-62-9, irreversible EGFR and Her2 inhibitor) , AEE-788
  • the inhibitor is selective for Her2 and exhibits little or no activity against other kinases.
  • Aurora Kinase Inhibitors that selectively decrease or block the expression of an Aurora kinase include, but are not limited to, antisense, siRNA and miRNA molecules.
  • siRNA inhibition of Aurora kinase is disclosed in, e.g., Tao, et al. (2007) Br. J. Cancer 97 ( 12 ) : 1664-1672 ; Umene, et al . (2015) Int. J. Oncol. 46 ( 4 ): 1498-1506.
  • Examples of small molecule inhibitors of Aurora kinase include, but are not limited to, SNS314 Mesylate (CAS 1146618-41-8, pan Aurora inhibitor, see US 2016/0287602, US 2015/0329828 and US 2011/0014191), PHA- 680632 (CAS 398493-79-3, pan Aurora inhibitor), VE-465 (Tozasertib, VX-680 or MK0457, CAS 639089-54-6), Barasertib (AZD1152, CAS 722544-51-6, Aurora B kinase inhibitor), Alisertib (MLN8237, CAS 1028486-01-2, Aurora A kinase inhibitor), Danusertib (PHA-739358, CAS 827318-97-8, pan Aurora inhibitor) , PF-03814735 (CAS 942487-16-3, dual Aurora A/B inhibitor), AMG 900 (CAS 945595-80-2, pan Aurora inhibitor), and derivatives and combinations thereof.
  • PDGFR Inhibitors that selectively decrease or block the expression of a PDGFR include, but are not limited to, antisense, siRNA and miRNA molecules.
  • siRNA inhibition of PDGFR is disclosed in, e.g., Chen, et al . (2008) liver Int. 28 (10) : 1446-1457; Kaulfuft, et al. (2013) Oncotarget 4 (7) : 1037-49; Yeh, et al . (2011) BMC Cancer 11:139.
  • Anti-PDGFR antibodies are also known in the art for use in modulating the activity of PDGFR.
  • Examples of anti- PDGFR antibodies include, but are not limited to, IMC-3G3 (anti-PDGFRa antibody; EP 2100618) and IMC-2C5 (PDGFR antibody; Shen, et al. (2009) Neoplasia 11 ( 6 ): 594-604 ) .
  • Examples of small molecule inhibitors of PDGFR include, but are not limited to, Kill502 (CAS 347155-76-4) , imatinib ( GLEEVEC/ST571 , CAS 220127-57-1, PDGFRa/BCR-ABL/c- kit inhibitor), Ponatinib (AP24534, CAS 943319-70-8, Abl/PDGFR /VEGFR2/FGFRl/Src inhibitor), Telatinib (CAS 332012-40-5, VEGFR/c-Kit /PDGFR inhibitor), Amuvatinib (MP- 470, CAS 850879-09-3, c-Kit /PDGFRa/Fit3 inhibitor), Crenolanib (CP-868596, CAS 670220-88-9, selective inhibitor of PDGFRa/ , see US 7,071,337, US 7,183,414, US 2015/0238479 and US 2010/0016353), Axitinib (CAS 319460
  • Atonal-associated factors are a family of transcription factors that transdifferentiate supporting cells into sensory hair cells in the ear. Atonal-associated factors are transcription factors of the basic helix-loop-helix (bHLH) family of proteins. The basic domain of the protein is responsible for DNA binding and function of the protein. The Drosophila bHLH protein (ato) activates genes associated with the development of sensory organs of the insect, specifically chordotonal organs.
  • bHLH basic helix-loop-helix
  • Atonal-associated factors also referred to as Atonal Homolog 1 (Atohl) proteins are found in a variety of animals and insects, including mice (mouse atonal homolog 1 (Mathl)), chickens (chicken atonal homolog 1 (Cathl) ) , Xenopus (Xenopus atonal homolog 1 (Xathl) ) , and humans (human atonal homolog 1 (Hathl) ) .
  • Mathl is highly homologous to ato in the bHLH domain (82% amino acid similarity) with 100% conservation of the basic domain, and functions in determining cell fate in mice. Mathl has been shown to be essential for hair cell development and can stimulate hair cell regeneration in the ear. Mathl is further characterized in, for example, Ben-Arie, et al .
  • Hathl is the human counterpart of Mathl.
  • the atonal-associated factor is Mathl or Hathl or a protein sharing significant amino acid sequence similarity with that of Mathl and Hathl. Atonal- associated factors are further described in WO 2000/73764.
  • a protein having significant amino acid sequence similarity with that of Mathl and Hathl desirably has an amino acid sequence that is at least about 50% identical to the amino acid sequence of Hathl (NP_005163) or Mathl (NP_031526), and has the ability to transdifferentiate supporting cells into sensory hair cells.
  • the Atonal-associated factor has at least about 60% amino acid sequence identity (e.g., at least about 65%, or at least about 70%, sequence identity), preferably at least about 75% amino acid sequence identity (e.g., at least about 80%, or at least about 85%, sequence identity) , and most preferably at least about 90% amino acid sequence identity (e.g., at least about 95% sequence identity) with the Hathl (NP_005163) or Mathl (NP_031526) amino acid sequence.
  • 60% amino acid sequence identity e.g., at least about 65%, or at least about 70%, sequence identity
  • at least about 75% amino acid sequence identity e.g., at least about 80%, or at least about 85%, sequence identity
  • amino acid sequence identity e.g., at least about 95% sequence identity
  • nucleic acid sequence encoding the Atonal-associated factor preferably the nucleic acid sequence encodes the Hathl (NP_005163) or Mathl (NP_031526) amino acid sequence (i.e., the portion of the Hathl or Mathl genes that encode the Hathl and Mathl proteins absent the regulatory sequences associated with the gene) or cDNA encoding the Hathl or Mathl protein.
  • Nucleic acid sequences encoding Hathl and Mathl are publicly available under GENBANK Accession Nos. NM_005172 and NM_031526, respectively.
  • Hathl or Mathl proteins and nucleic acids are particularly useful, many modifications and variations (e.g., mutation) of the Hathl or Mathl sequences are possible and appropriate in the context of the invention.
  • the degeneracy of the genetic code allows for the substitution of nucleotides throughout the coding sequence, as well as in the translational stop signal, without alteration of the encoded polypeptide.
  • substitutions can be deduced from the known amino acid sequence of an atonal-associated factor or nucleic acid sequence encoding an atonal-associated factor and can be constructed by conventional synthetic or site-specific mutagenesis procedures. Synthetic DNA methods can be carried out in accordance with the procedures of Itakura, et al.
  • nucleic acid sequence can encode an atonal-associated peptide with extensions on either the N- or C-terminus of the protein, so long as the resulting atonal-associated factor retains activity (i.e., the ability to transdifferentiate supporting cells into sensory hair cells) .
  • Atonal-associated factors is dependent on the helix-loop-helix (HLH) portion of the protein, particularly the basic region of the HLH domain (Chien, et al. (1996) Proc. Natl. Acad. Sci. 93:13239- 13244), which includes the amino acid sequence AANARERRRMHGLNHAFDQLR (SEQ ID NO : 1 ) . Accordingly, any modification of the atonal-associated factor amino acid sequence desirably is located outside of the basic domain of the protein. Exemplary constructs and expression vectors harboring nucleic acids encoding atonal-associated factor are provided WO 2004/076626.
  • the invention provides for the modulation of sensory perception in an animal by administering to the inner ear an inhibitor of EGFR signaling and optionally an expression vector (e.g., expression viral vector) harboring a nucleic acid molecule encoding an atonal-associated factor and/or otoprotective agent.
  • an expression vector e.g., expression viral vector
  • modulating sensory perception it is meant achieving, at least in part, the ability to recognize and adapt to environmental changes.
  • modulation in sensory perception is associated with the generation or protection of sensory hair cells that convert mechanical stimuli in the inner ear into neural impulses, which are then processed in the brain such that an animal is aware of environmental change, e.g., sound, language, or body/head position.
  • Sensory hair cells are preferably generated in the organ of Corti and/or vestibular apparatus.
  • sensory hair cells which would otherwise be initially or further damaged or lost due to, e.g., ototoxic agents, are protected from damage or loss by the administration of an inhibitor of EGFR signaling and optionally an otoprotective agent.
  • the combination of the inhibitor of EGFR signaling and the nucleic acid molecule for expressing an atonal-associated factor increases reprogramming efficiency and/or terminal differentiation of some reprogrammed cells thereby facilitating the generation of sensory hair cells that allow perception (or recognition) of stimuli in the inner ear.
  • Sensory hair cell generation can be determined using a variety of means, such as those known to one skilled in the art. Hair cells can be detected via scanning electron microscopy and/or via detection of myosin Vila, a hair cell-specific protein detected by immunochemistry . However, the mere presence of sensory hair cells does not necessarily imply a functional system for recognizing environmental stimuli. Functional sensory hair cells must be operably linked to neural pathways, such that mechanical stimuli are translated to nerve impulses recognized by the brain. Accordingly, while detection of hair cell generation is appropriate for determining successful expression of the atonal-associated nucleic acid seguence to target tissue, examination of subject awareness is a more desirable indicator of changes in sensory perception.
  • a change in the ability of a subject to detect sound is readily accomplished through administration of simple hearing tests, such as a tone test commonly administered by an audiologist.
  • simple hearing tests such as a tone test commonly administered by an audiologist.
  • a reaction to different frequencies indicates a change in sensory perception.
  • comprehension of language also is appropriate. For example, it is possible for a subject to hear while being unable to understand speech.
  • a change in perception is indicated by the ability to distinguish different types of acoustic stimuli, such as differentiating language from background noise, and by understanding speech. Speech threshold and discrimination tests are useful for such evaluations .
  • a baseline value is recorded prior to the inventive method using any appropriate sensory test.
  • a subject is reevaluated at an appropriate time period following the inventive method (e.g., 1 hour, 6 hours, 12 hours, 18 hours, 1 day, 3 days, 5 days, 7 days, 14 days, 21 days, 28 days, 2 months, 3 months or more following the inventive method) , the results of which are compared to baseline results to determine a change in sensory perception.
  • the inventive method promotes the protection and/or generation of sensory hair cells that allow perception of stimuli. Accordingly, this invention provides a method for the prevention, treatment, control, amelioration, or reduction of risk of hearing impairments, loss and disorders by administering to a subject in need of treatment an inhibitor of EGFR signaling and optionally an expression vector (e.g., expression viral vector) harboring a nucleic acid molecule encoding an atonal-associated factor and/or one or more otoprotective/regenerative agents. Ideally, the inventive method prophylactically or therapeutically treats an animal for at least one disorder associated with loss, damage, absence of sensory hair cells, such as hearing loss and balance disorders.
  • an expression vector e.g., expression viral vector
  • Hearing loss can be caused by damage of hair cells of the organ of Corti due to bacterial or viral infection, heredity, physical injury, acoustic trauma, ototoxic drugs ⁇ e.g., aminoglycoside antibiotic or cisplatin) and the like. While hearing loss is easily identified, balance disorders manifest in a broad variety of complications easily attributable to other ailments. Symptoms of a balance disorder include disorientation, dizziness, vertigo, nausea, blurred vision, clumsiness, and frequent falls. Balance disorders treated by the inventive method preferably involve a peripheral vestibular disorder ⁇ i.e., a disturbance in the vestibular apparatus) involving dysfunctional translation of mechanical stimuli into neural impulses due to damage or lack of sensory hair cells.
  • a peripheral vestibular disorder ⁇ i.e., a disturbance in the vestibular apparatus
  • a subject in need of treatment is administered an effective amount of inhibitor of EGFR signaling.
  • the inhibitor of EGFR signaling inhibits the expression or activity of EGFR, a Ras/Raf/MEK/ERK/MAPK protein, a JAK/S AT protein, a PI3K/AKT/mT0R protein, a NCK-PAK-JNK protein, a PLC-DAG-PKC protein, or a cell cycle-associated protein kinase associated with or downstream of EGFR.
  • prevention of hearing loss is achieved by administering to a subject in need of treatment an inhibitor of a cell cycle-associated protein kinase associated with or downstream of EGFR.
  • prevention of hearing loss is achieved by administering to a subject in need of treatment an inhibitor of Her-2, Aurora kinase, B-Raf, or PDGFR expression or activity.
  • the inhibitor of EGFR signaling can be administered alone or in combination with one or more otoprotective agents.
  • otoprotective agent refers to an agent that reduces or prevents noise-induced hearing loss, chemically-induced hearing loss, or age-induced hearing impairment or otherwise protects against hearing impairment.
  • otoprotective agents include, but are not limited to, PARP-1 inhibitors; pirenzepine LS-75, otenzepad, AQ-RA741, viramune, BIBN 99, DIBD, telenzepine (see US 2011/0263574); methionine (see US 7,071,230); IGF- 1, FGF-2, aspirin, reduced glutathione, N-methyl- ( D) - glucaminedithiocarbamate, and iron chelators such as tartrate and maleate. See also US 2005/0101534 for additional otoprotective agents.
  • a subject in need of treatment is administered an effective amount of inhibitor of EGFR signaling in combination with an expression vector harboring a nucleic acid molecule for expressing an atonal-associated factor.
  • the inhibitor of EGFR signaling inhibits the expression or activity of EGFR, a Ras/Raf/MEK/ERK/MAPK protein, a JAK/STAT protein, a PI3K/AKT/mTOR protein, a NCK-PAK-JNK protein, a PLC-DAG-PKC protein, or a cell cycle-associated protein kinase associated with or downstream of EGFR.
  • treatment of hearing loss is achieved by administering to a subject in need of treatment an inhibitor of EGFR, a Ras/Raf/ EK/ERK/MAPK protein, a JAK/STAT protein, a PI3K/AKT/mTOR protein, a NCK-PAK-JNK protein, or a PLC-DAG-PKC protein.
  • treatment of hearing loss is achieved by administering to a subject in need of treatment an inhibitor of EGFR, PLC, STAT3, JAK2, PI3K or MEK expression or activity.
  • the inhibitor of EGFR signaling and expression vector harboring a nucleic acid molecule for expressing an atonal-associated factor can be administered alone or in combination with one or more regenerative agents.
  • regenerative agent refers to an agent that stimulates or promotes sensory hair cell regeneration.
  • regenerative agents include, but are not limited to, nicotinamide riboside (see US 2015/0174148); siRNA targeting Hesl (see US 9,101,647); PKA inhibitors (see US 6,268,351); a Myc family protein such as c-Myc, N-Myc or L-Myc (see US 2015/0079110) ; and ellipticine derivatives and/or a GSK-3 inhibitor (see US 9, 370, 510) .
  • Protection against and prevention or treatment of hearing loss or impairment can be in the context of conditions including, but not limited to, tinnitus, ringing, Presbyacusis , auditory neuropathy, acoustic trauma, acoustic neuroma, Pendred syndrome, Usher syndrome, Wardenburg syndrome, non-syndromic sensorineural deafness, otitis media, otosclerosis, Meniere's disease, ototoxicity, labyrinthitis, as well as hearing impairments caused by infection (i.e., measles, mumps, or meningitis), medicines such as antibiotics, and some cancer treatments (i.e., chemotherapy and radiation therapy) .
  • infections i.e., measles, mumps, or meningitis
  • medicines such as antibiotics
  • cancer treatments i.e., chemotherapy and radiation therapy
  • the hearing impairment is drug-induced.
  • the drug is a chemotherapeutic agent. More specifically, the drug is a platinum-based chemotherapeutic agent such as carboplatin, cisplatin, transplatin, nedaplatin, oxaliplatin, picoplatin, satraplatin, transplatin, and triplatin, or a pharmaceutically acceptable salt thereof.
  • the platinum-based chemotherapeutic agent is cisplatin, or a pharmaceutically acceptable salt thereof.
  • the drug is an antibiotic, including, but not limited to, daunorubicin, doxorubicin, epirubicin, idarubicin, actinomycin-D, bleomycin, mitomycin-C, amikacin, apramycin, arbekacin, astromicin, bekanamycin, dibekacin, framycetin, gentamicin, hygromycin B, isepamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin, and verdamicin, or a pharmaceutically acceptable salt thereof.
  • an antibiotic including, but not limited to, daunorubicin, doxorubicin, epirubicin, idarubicin, actinomycin-D, bleomycin, mitomycin-C, amikacin, apramycin,
  • the hearing impairment is age- related, noise-induced or a balance or orientation-related disorder.
  • balance disorders include, but are not limited to, induced or spontaneous vertigo, dysequilibrium, increased susceptibility to motion sickness, nausea, vomiting, ataxia, labyrinthitis, oscillopsia, nystagmus, syncope, lightheadedness, dizziness, increased falling, difficulty walking at night, Meniere's disease, and difficulty in visual tracking and processing.
  • the noise-induced hearing loss may be temporary or permanent.
  • TBI is often accompanied by a diverse range of disruption or damage to the auditory sensory system, which is highly vulnerable to blast injury. Extreme physical blast force can cause damage of various types to the peripheral auditory system, including rupture of the tympanic membrane (TM, eardrum) , fracture of the middle ear bones, dislocation of sensory hair cells from the basilar membrane, and loss of spiral ganglia that innervate hair cells.
  • TM tympanic membrane
  • TM tympanic membrane
  • the inhibitor of EGFR signaling and optional expression vector harboring a nucleic acid molecule for expressing an atonal-associated factor can be administered locally, e.g., to the inner ear of the subject.
  • the inhibitor of EGFR signaling and optional expression vector harboring a nucleic acid molecule for expressing an atonal-associated factor can be administered systemically .
  • the inhibitor of EGFR signaling and optional expression vector harboring a nucleic acid molecule for expressing an atonal-associated factor can be administered via injection into one or more of the scala tympani, cochlear duct, scala vestibule of the cochlea, into the auditory nerve trunk in the internal auditory meatus, or into the middle ear space across the transtympanic membrane/ear drum.
  • the EGFR signaling and expression vector harboring a nucleic acid molecule for expressing an atonal-associated factor can be administered via the same or different routes.
  • the disclosed molecules can be used in combination with one or more other drugs in the treatment, prevention, control, amelioration, or reduction of risk of hearing impairments and disorders for which disclosed molecules or the other drugs can have utility, where the combination of the drugs together are safer or more effective than either drug alone.
  • Such other drug(s) can be administered, by a route and in an amount commonly used therefor, contemporaneously or sequentially with a compound of the present invention.
  • a pharmaceutical composition in unit dosage form containing such other drugs and a disclosed compound is preferred.
  • the combination therapy can also include therapies in which a disclosed molecule and one or more other drugs are administered on different overlapping schedules. It is also contemplated that when used in combination with one or more other active ingredients, the disclosed molecules and the other active ingredients can be used in lower doses than when each is used singly.
  • the methods herein are useful in the prevention or treatment of both acute and persistent, progressive disorders associated with lack of or damage to functional sensory hair cells.
  • the drugs herein can be administered using a single application or multiple applications within a short time period.
  • persistent diseases such as hearing loss, or disorders stemming from a massive loss of sensory hair cells, numerous rounds of administration of the drugs herein may be necessary to realize a therapeutic effect.
  • the subject e.g., human or other animal
  • Subjects benefiting from treatment include those at risk of hair cell loss and/or a patient with hair cell loss.
  • a subject having or at risk for developing a hearing loss can hear less well than the average subject (e.g., an average human being), or less well than a subject before experiencing the hearing loss.
  • hearing can be diminished by at least 5%, 10%, 30%, 50% or more.
  • Methods for measuring hearing are well- known and include pure tone audiometry, air conduction, and bone conduction tests. These exams measure the limits of loudness (intensity) and pitch (frequency) that a human can hear.
  • Hearing tests in humans include behavioral observation audiometry (for infants to seven months), visual reinforcement orientation audiometry (for children 7 months to 3 years) and play audiometry for children older than 3 years.
  • Oto-acoustic emission testing can be used to test the functioning of the cochlear hair cells
  • electro-cochleography provides information about the functioning of the cochlea and the first part of the nerve pathway to the brain.
  • treatment can be continued with or without modification or can be stopped.
  • the methods described herein can be used to generate hair cell growth in the ear and/or to increase the number of hair cells in the ear (e.g., in the inner, middle, and/or outer ear) .
  • an effective amount of a molecule described herein is an amount that increases the number of hair cells in the ear by about 2-, 3-, 4-, 6-, 8-, or 10-fold, or more, as compared to the number of hair cells before treatment.
  • This new hair cell growth can effectively restore or establish at least a partial improvement in the subject's ability to hear.
  • administration of a stimulatory agent and an inhibitory agent of this invention can improve hearing loss by about 5, 10, 15, 20, 40, 60, 80, 100% or more .
  • Expression Vectors are suitable for introducing a nucleic acid sequence to the inner ear.
  • suitable expression vectors include, for instance, plasmids, plasmid-liposome complexes, and viral vectors, e.g., parvoviral-based vectors (i.e., adeno-associated virus
  • AAV adenoviral chimeric vectors
  • retroviral vectors retroviral vectors
  • HSV herpes simplex virus
  • AAV-adenoviral chimeric vectors AAV-adenoviral chimeric vectors
  • adenovirus-based vectors Any of these expression vectors can be prepared using standard recombinant DNA techniques described in, e.g., Sambrook, et al . (1989) Molecular Cloning, A Laboratory Manual, 2 nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY; and Ausubel, et al .
  • Plasmids genetically engineered circular double- stranded DNA molecules, can be designed to contain an expression cassette for delivery of a nucleic acid sequence to the inner ear.
  • plasmids were the first vector described for the administration of therapeutic nucleic acids, the level of transfection efficiency is poor compared with other techniques .
  • liposomes By complexing the plasmid with liposomes, the efficiency of gene transfer in general is improved. While the liposomes used for plasmid-mediated gene transfer strategies have various compositions, they are typically synthetic cationic lipids. Advantages of plasmid-liposome complexes include their ability to transfer large pieces of DNA encoding a therapeutic nucleic acid and their relatively low immunogenicity .
  • Plasmids also can be modified to prolong transgene expression as described in US 6,165,754. Expression of a transgene in the ear using plasmids has been described (see, for example, Jero, et al . (2001) Human Gene Ther . 12:539-549). While plasmids are suitable for use in the inventive method, preferably the expression vector is a viral vector.
  • AAV vectors are viral vectors of particular interest for use in gene therapy protocols.
  • AAV is a DNA virus, which is not known to cause human disease.
  • AAV requires co- infection with a helper virus (i.e., an adenovirus or a herpes virus), or expression of helper genes, for efficient replication.
  • helper virus i.e., an adenovirus or a herpes virus
  • AAV vectors used for administration of a therapeutic nucleic acid have approximately 96% of the parental genome deleted, such that only the terminal repeats (ITRs) , which contain recognition signals for DNA replication and packaging, remain. This eliminates immunologic or toxic side effects due to expression of viral genes.
  • Host cells containing an integrated AAV genome show no change in cell growth or morphology (see, for example, US 4,797,368). Although efficient, the need for helper virus or helper genes can be an obstacle for widespread use of this vector.
  • Retrovirus is an RNA virus capable of infecting a wide variety of host cells. Upon infection, the retroviral genome integrates into the genome of its host cell and is replicated along with host cell DNA, thereby constantly producing viral RNA and any nucleic acid sequence incorporated into the retroviral genome.
  • pathogenic retroviruses e.g., human immunodeficiency virus
  • retroviral vector can additionally be manipulated to render the virus replication-incompetent.
  • retroviral vectors are thought to be particularly useful for stable gene transfer in vivo.
  • Lentiviral vectors such as HIV-based vectors, are exemplary of retroviral vectors used for gene delivery. Unlike other retroviruses, HIV-based vectors are known to incorporate their passenger genes into non-dividing cells and, therefore, are particularly useful in the sensory epithelium of the inner ear where sensory cells do not regenerate .
  • HSV-based viral vectors are suitable for use as an expression vector to introduce nucleic acids into the inner ear for transduction of target cells.
  • the mature HSV virion is composed of an enveloped icosahedral capsid with a viral genome composed of a linear double-stranded DNA molecule that is 152 kb.
  • Most replication-deficient HSV vectors contain a deletion to remove one or more intermediate-early genes to prevent replication.
  • Advantages of the herpes vector are its ability to enter a latent stage that can result in long-term DNA expression, and its large viral DNA genome that can accommodate exogenous DNA up to 25 kb. Of course, this ability is also a disadvantage in terms of short-term treatment regimens.
  • HSV- based vectors appropriate for use in the inventive methods, see, for example, US 5,837,532, US 5,846,782, US 5,849,572, US 5,804,413, WO 1991/02788, WO 1996/04394, WO 1998/15637, and WO 1999/06583.
  • Adenovirus is a 36-kb double-stranded DNA virus that efficiently transfers DNA in vivo to a variety of different target cell types.
  • the virus is preferably made replication-deficient by deleting select genes required for viral replication.
  • the expendable non-replication-essential E3 region is also frequently deleted to allow additional room for a larger DNA insert.
  • the vector can be produced in high titers and can efficiently transfer DNA to replicating and non- replicating cells. Genetic information transferred to a cell by way of an adenoviral vector remains epi- chromosomal, thus eliminating the risks of random insertional mutagenesis and permanent alteration of the genotype of the target cell.
  • the integrative properties of AAV can be conferred to adenovirus by constructing an AAV-Ad chimeric vector.
  • the AAV inverted terminal repeats (ITRs) and nucleic acid encoding the Rep protein incorporated into an adenoviral vector enables the adenoviral vector to integrate into a mammalian cell genome. Therefore, AAV-Ad chimeric vectors are an interesting option for use in the context of the invention.
  • the expression vector of the inventive method is a viral vector, more preferably, the expression vector is an adenoviral vector.
  • Adenovirus from any origin, any subtype, mixture of subtypes, or any chimeric adenovirus can be used as the source of the viral genome for the adenoviral vector of the invention.
  • a human adenovirus preferably is used as the source of the viral genome for the replication-deficient adenoviral vector.
  • the adenovirus can be of any subgroup or serotype.
  • an adenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42- 48), subgroup E (e.g., serotype 4), subgroup F (e.g., serotypes 40 and 41), an unclassified serogroup (e.g., serotypes 49 and 51), or any other adenoviral serotype.
  • subgroup A e.g., serotypes 12, 18, and 31
  • subgroup B e.g., serotypes 3, 7, 11, 14, 16, 21, 34, 35, and 50
  • subgroup C e.g., serotypes 1, 2, 5, and 6
  • subgroup D e.g., serotypes
  • Adenoviral serotypes 1 through 51 are available from the American Type Culture Collection (ATCC, Manassas, VA) .
  • the adenoviral vector is of subgroup C, especially serotype 2 or even more desirably serotype 5.
  • non-group C adenoviruses can be used to prepare replication- deficient adenoviral gene transfer vectors for delivery of DNA to target cells in the inner ear.
  • Preferred adenoviruses used in the construction of non-group C adenoviral gene transfer vectors include Adl2 (group A) , Ad7 and Ad35 (group B) , Ad30 and Ad36 (group D) , Ad4 (group E) , and Ad41 (group F) .
  • Non-group C adenoviral vectors methods of producing non-group C adenoviral vectors, and methods of using non-group C adenoviral vectors are disclosed in, for example, US 5,801,030, US 5,837,511, US 5,849,561, WO 1997/12986 and WO 1998/53087.
  • Preferred non- human adenoviruses include, but are not limited to, simian
  • SAV 25 bovine, canine, porcine adenoviruses.
  • the adenoviral vector is preferably replication- deficient.
  • replication-deficient is meant that the adenoviral vector comprises an adenoviral genome that lacks at least one replication-essential gene function (i.e., such that the adenoviral vector does not replicate in typical host cells, especially those in the human patient that could be infected by the adenoviral vector in the course of treatment in accordance with the invention) .
  • a deficiency in a gene, gene function, or gene or genomic region, as used herein, is defined as a deletion of sufficient genetic material of the viral genome to impair or obliterate the function of the gene whose nucleic acid sequence was deleted in whole or in part.
  • Replication-essential gene functions are those gene functions that are required for replication (e.g., propagation) and are encoded by, for example, the adenoviral early regions (e.g., the El, E2, and E4 regions), late regions (e.g., the L1-L5 regions), genes involved in viral packaging (e.g., the IVa2 gene), and virus-associated RNAs (e.g., VA-RNA1 and/or VA-RNA-2). More preferably, the replication-deficient adenoviral vector comprises an adenoviral genome deficient in at least one replication-essential gene function of one or more regions of the adenoviral genome.
  • the adenoviral vector is deficient in at least one gene function of the El region or the E4 region of the adenoviral genome required for viral replication (denoted an El-deficient adenoviral vector or an E4-deficient adenoviral vector) .
  • the recombinant adenovirus also can have a mutation in the major late promoter (LP) , as discussed in WO 2000/00628.
  • the adenoviral vector is deficient in at least one replication-essential gene function (desirably all replication-essential gene functions) of the El region and at least part of the nonessential E3 region (e.g., an Xbal deletion of the E3 region) (denoted an El/E3-deficient adenoviral vector) .
  • the adenoviral vector can be deficient in part or all of the ElA region and part or all of the E1B region, e.g., in at least one replication- essential gene function of each of the ElA and E1B regions.
  • the adenoviral vector When the adenoviral vector is deficient in at least one replication-essential gene function in one region of the adenoviral genome (e.g., an El- or El/E3-deficient adenoviral vector) , the adenoviral vector is referred to as "singly replication-deficient . "
  • the adenoviral vector of the invention can be "multiply replication-deficient,” meaning that the adenoviral vector is deficient in one or more replication- essential gene functions in each of two or more regions of the adenoviral genome.
  • the aforementioned El- deficient or El/E3-deficient adenoviral vector can be further deficient in at least one replication-essential gene function of the E4 region (denoted an E1/E4- or El/E3/E4-deficient adenoviral vector), and/or the E2 region (denoted an E1/E2- or El/E2/E3-deficient adenoviral vector) , preferably the ⁇ 2 ⁇ region (denoted an E1/E2A- or El/E2A/E3-deficient adenoviral vector) .
  • the adenoviral vector lacks replication-essential gene functions of only those replication-essential gene functions encoded by the early regions of the adenoviral genome, although this is not required in all contexts of the invention.
  • a preferred multiply-deficient adenoviral vector comprises an adenoviral genome having deletions of nucleotides 457-3332 of the El region, nucleotides 28593- 30470 of the E3 region, nucleotides 32826-35561 of the E4 region, and, optionally, nucleotides 10594-10595 of the region encoding VA-RNAl . However, other deletions may be appropriate.
  • Nucleotides 356-3329 or 356-3510 can be removed to create a deficiency in replication-essential El gene functions.
  • Nucleotides 28594-30469 can be deleted from the E3 region of the adenoviral genome. While the specific nucleotide designations recited above correspond to the adenoviral serotype 5 genome, the corresponding nucleotides for non-serotype 5 adenoviral genomes can easily be determined by those of ordinary skill in the art.
  • the adenoviral vector when multiply replication- deficient, especially in replication-essential gene functions of the El and E4 regions, preferably includes a spacer element to provide viral growth in a complementing cell line similar to that achieved by singly replication- deficient adenoviral vectors, particularly an El-deficient adenoviral vector.
  • the spacer element can contain any sequence or sequences which are of a desired length, such as sequences at least about 15 base pairs (e.g., between about 15 base pairs and about 12, 000 base pairs), preferably about 100 base pairs to about 10,000 base pairs, more preferably about 500 base pairs to about 8,000 base pairs, even more preferably about 1,500 base pairs to about 6,000 base pairs, and most preferably about 2,000 to about 3,000 base pairs in length.
  • the spacer element sequence can be coding or non-coding and native or non-native with respect to the adenoviral genome, but does not restore the replication-essential function to the deficient region.
  • the use of a spacer in an adenoviral vector is described in US 5,851,806.
  • the replication-deficient or conditionally-replicating adenoviral vector is an El/E4-deficient adenoviral vector wherein the L5 fiber region is retained, and a spacer is located between the L5 fiber region and the right-side ITR.
  • the E4 poiyadenylation sequence alone or, most preferably, in combination with another sequence, exists between the L5 fiber region and the right-side ITR, so as to sufficiently separate the retained L5 fiber region from the right-side ITR, such that viral production of such a vector approaches that of a singly replication-deficient adenoviral vector, particularly an El-deficient adenoviral vector.
  • the adenoviral vector can be deficient in replication-essential gene functions of only the early regions of the adenoviral genome, only the late regions of the adenoviral genome, and both the early and late regions of the adenoviral genome.
  • the adenoviral vector also can have essentially the entire adenoviral genome removed, in which case it is preferred that at least either the viral ITRs and one or more promoters or the viral ITRs and a packaging signal are left intact (i.e., an adenoviral amplicon) .
  • the 5' or 3' regions of the adenoviral genome comprising ITRs and packaging sequence need not originate from the same adenoviral serotype as the remainder of the viral genome.
  • the 5' region of an adenoviral serotype 5 genome i.e., the region of the genome 5' to the adenoviral El region
  • the corresponding region of an adenoviral serotype 2 genome e.g., the Ad5 genome region 5' to the El region of the adenoviral genome is replaced with nucleotides 1-456 of the Ad2 genome
  • Suitable replication-deficient adenoviral vectors including multiply replication-deficient adenoviral vectors, are disclosed in US 5,837,511, US 5,851,806, US 5,994,106, US 2001/0043922, US 2002/0004040, US 2002/0031831, US 2002/0110545, WO 1995/34671, WO 1997/12986, and WO 1997/21826.
  • the replication- deficient adenoviral vector is present in a pharmaceutical composition virtually free of replication-competent adenovirus (RCA) contamination (e.g., the pharmaceutical composition comprises less than about 1% of RCA contamination) .
  • the pharmaceutical composition is RCA-free.
  • Adenoviral vector compositions and stocks that are RCA-free are described in US 5,944,106, US 6,482,616, US 2002/0110545 and WO 1995/34671.
  • the expression vector of the inventive method is a multiply replication- deficient adenoviral vector lacking all or part of the El region, all or part of the E3 region, all or part of the E4 region, and, optionally, all or part of the E2 region. It is believed that multiply deficient vectors are particularly suited for delivery of exogenous nucleic acid sequences to the ear.
  • Adenoviral vectors deficient in at least one replication-essential gene function of the El region are most commonly used for gene transfer in vivo.
  • currently used singly replication-deficient adenoviral vectors can be detrimental to the sensitive cells of the epithelium of the inner ear, causing damage to the very cells to be treated.
  • Adenoviral vectors that are deficient in at least one replication-essential gene function of the E4 region are less toxic to cells than El- deficient adenoviral vectors (see, for example, Wang, et al. (1996) Nature Med. 2(6):714-716 and US 6,228,646). Accordingly, damage to existing hair cells and supporting cells can be minimized by employing an El, E4-deficient adenoviral vector to deliver the nucleic acid sequence encoding the atonal-associated factor to inner ear cells.
  • an at least E4-deficient adenoviral vector expresses a transgene at high levels for a limited amount of time in vivo and that persistence of expression of a transgene in a at least E4-deficient adenoviral vector can be modulated through the action of a trans-acting factor, such as HSV ICPO, Ad pTP, CMV-IE2, CMV-IE86, HIV tat, HTLV-tax, HBV-X, AAV Rep 78, the cellular factor from the U205 osteosarcoma cell line that functions like HSV ICPO, or the cellular factor in PC12 cells that is induced by nerve growth factor, among others.
  • a trans-acting factor such as HSV ICPO, Ad pTP, CMV-IE2, CMV-IE86, HIV tat, HTLV-tax, HBV-X, AAV Rep 78, the cellular factor from the U205 osteosarcoma cell line that functions like HSV ICPO, or the cellular factor
  • the multiply deficient adenoviral vector e.g., the at least E4- deficient adenoviral vector
  • a second expression vector comprises a nucleic acid sequence encoding a trans-acting factor that modulates the persistence of expression of the nucleic acid sequence encoding the atonal-associated factor, as described in, for example, US 6,225,113, US 6,660,521, US 6,649,373, and WO 2000/34496.
  • Replication-deficient adenoviral vectors are typically produced in complementing cell lines that provide gene functions not present in the replication-deficient adenoviral vectors, but required for viral propagation, at appropriate levels in order to generate high titers of viral vector stock.
  • a preferred cell line complements for at least one and preferably all replication-essential gene functions not present in a replication-deficient adenovirus.
  • the complementing cell line can complement for a deficiency in at least one replication-essential gene function encoded by the early regions, late regions, viral packaging regions, virus-associated RNA regions, or combinations thereof, including all adenoviral functions
  • the complementing cell line complements for a deficiency in at least one replication-essential gene function (e.g., two or more replication-essential gene functions) of the El region of the adenoviral genome, particularly a deficiency in a replication-essential gene function of each of the E1A and E1B regions.
  • the complementing cell line can complement for a deficiency in at least one replication-essential gene function of the E2 (particularly as concerns the adenoviral DNA polymerase and terminal protein) and/or E4 regions of the adenoviral genome.
  • a cell that complements for a deficiency in the E4 region comprises the E4-ORF6 gene sequence and produces the E4-ORF6 protein.
  • Such a cell desirably comprises at least ORF6 and no other ORF of the E4 region of the adenoviral genome.
  • the cell line preferably is further characterized in that it contains the complementing genes in a non-overlapping fashion with the adenoviral vector, which minimizes, and practically eliminates, the possibility of the vector genome recombining with the cellular DNA. Accordingly, the presence of replication competent adenoviruses (RCA) is minimized if not avoided in the vector stock, which, therefore, is suitable for certain therapeutic purposes, especially gene therapy purposes.
  • RCA replication competent adenoviruses
  • Complementing cell lines for producing the adenoviral vector include, but are not limited to, 293 cells (see, e.g., Graham, et al. (1977) J. Gen. Virol. 36:59-72), PER.C6 cells (see, e.g., WO 1997/00326, US 5,994,128 and US 6,033,908), and 293-ORF6 cells (see, e.g., WO 1995/34671 and Brough, et al . (1997) J. Virol. 71:9206- 9213) . In some instances, the complementing cell will not complement for all required adenoviral gene functions .
  • Helper viruses can be employed to provide the gene functions in trans that are not encoded by the cellular or adenoviral genomes to enable replication of the adenoviral vector.
  • Adenoviral vectors can be constructed, propagated, and/or purified using the materials and methods set forth, for example, in US 5,965,358, US 5,994,128, US 6,033,908, US 6,168,941, US 6,329,200, US 6,383,795, US 6,440,728, US 6,447,995, US 6,475,757, US 2002/0034735, WO 1998/53087, WO 1998/56937, WO 1999/15686, WO 1999/54441, WO 2000/12765, WO 2001/77304, and WO 2002/29388, as well as the other references identified herein.
  • Non-group C adenoviral vectors including adenoviral serotype 35 vectors, can be produced using the methods set forth in, for example, US 5,837,511 US 5,849,561, WO 1997/12986 and WO 1998/53087. Moreover, numerous adenoviral vectors are available commercially .
  • the adenoviral vector's coat protein can be modified so as to decrease the adenoviral vector's ability or inability to be recognized by a neutralizing antibody directed against the wild-type coat protein. Such modifications are useful for multiple rounds of administration.
  • the coat protein of the adenoviral vector can be manipulated to alter the binding specificity or recognition of the adenoviral vector for a viral receptor on a potential host cell. Such manipulations can include deletion or substitution of regions of the fiber, penton, hexon, pllla, pVI, and/or pIX, insertions of various native or non-native ligands into portions of the coat protein, and the like.
  • Manipulation of the coat protein can broaden the range of ceils infected by the adenoviral vector or enable targeting of the adenoviral vector to a specific cell type.
  • the ability of an adenoviral vector to recognize a potential host cell can be modulated without genetic manipulation of the coat protein, i.e., through use of a bi-specific molecule. For instance, complexing an adenovirus with a bispecific molecule comprising a penton base- or fiber-binding domain and a domain that selectively binds a particular cell surface binding site enables the targeting of the adenoviral vector to a particular cell type.
  • the adenoviral capsid is modified to display a non-native amino acid sequence.
  • the non-native amino acid sequence can be inserted into or in place of an internal coat protein sequence (e.g., within an exposed loop of an adenoviral fiber protein) or fused to the terminus of an adenoviral coat protein (e.g., fused to the C-terminus of an adenoviral fiber protein, optionally using a linker or spacer sequence) .
  • the non-native amino acid sequence can be conjugated to any of the adenoviral coat proteins to form a chimeric coat protein.
  • the non-native amino acid sequence of the invention can be conjugated to, inserted into, or attached to a fiber protein, a penton base protein, a hexon protein, proteins IX, VI, or Ilia, etc.
  • a fiber protein e.g., a penton base protein
  • a hexon protein e.g., proteins IX, VI, or Ilia
  • sequences of such proteins , and methods for employing them in recombinant proteins are well known in the art (see, e.g., US
  • the coat protein portion of the chimeric coat protein can be a full-length adenoviral coat protein to which the ligand domain is appended, or it can be truncated, e.g., internally or at the C- and/or N- terminus .
  • the coat protein portion need not, itself, be native to the adenoviral vector.
  • the ligand is attached to the fiber protein, preferably it does not disturb the interaction between viral proteins or fiber monomers.
  • the non-native amino acid sequence preferably is not itself an oligomerization domain, as such can adversely interact with the trimerization domain of the adenovirus fiber.
  • the ligand is added to the virion protein, and is incorporated in such a manner as to be readily exposed to the substrate (e.g., at the N- or C-terminus of the protein, attached to a residue facing the substrate, positioned on a peptide spacer to contact the substrate, etc.) to maximally present the non-native amino acid sequence to the substrate.
  • the non-native amino acid sequence is incorporated into an adenoviral fiber protein at the C-terminus of the fiber protein (and attached via a spacer) or incorporated into an exposed loop (e.g., the HI loop) of the fiber to create a chimeric coat protein.
  • the non-native amino acid sequence is attached to or replaces a portion of the penton base, preferably it is within the hypervariable regions to ensure that it contacts the substrate.
  • the non-native amino acid sequence is attached to the hexon, preferably it is within a hypervariable region (Miksza, et al . (1996) J. Virol. 70(3) : 1836-44) .
  • a spacer sequence to extend the non-native amino acid sequence away from the surface of the adenoviral particle can be advantageous in that the non-native amino acid sequence can be more available for binding to a receptor and any steric interactions between the non-native amino acid sequence and the adenoviral fiber monomers is reduced.
  • a chimeric viral coat protein comprising a non- native ligand is desirably able to direct entry into cells of the viral, i.e., adenoviral, vector comprising the coat protein that is more efficient than entry into cells of a vector that is identical except for comprising a wild-type viral coat protein rather than the chimeric viral coat protein.
  • the chimeric virus coat protein binds a novel endogenous binding site present on the cell surface that is not recognized, or is poorly recognized by a vector comprising a wild-type coat protein.
  • the adenoviral capsid proteins can be altered to reduce or ablate binding to native adenoviral receptors (i.e., receptors bound by wild-type adenovirus) .
  • the portion of the adenoviral fiber protein which interacts with the coxsackie and adenovirus receptor (CAR) can be mutated by deletion, substitution, repositioning within the fiber protein, etc., such that the adenoviral fiber protein does not bind CAR.
  • the portion of the adenoviral penton protein that interacts with integrins can be altered to ablate native integrin binding.
  • the native binding sites located on adenoviral coat proteins which mediate cell entry are absent or disrupted. Two or more of the adenoviral coat proteins are believed to mediate attachment to cell surfaces (e.g., the fiber and penton base) .
  • Any suitable technique for altering native binding to a host cell e.g., a mesothelial cell or hepatocyte
  • exploiting differing fiber lengths to ablate native binding to cells can be accomplished via the addition of a binding sequence to the penton base or fiber knob.
  • the adenoviral fiber protein can be modified to reduce the number of amino acids in the fiber shaft, thereby creating a "short-shafted" fiber (as described in, for example, US 5,962,311).
  • the fiber proteins of some adenoviral serotypes are naturally shorter than others, and these fiber proteins can be used in place of the native fiber protein to reduce native binding of the adenovirus to its native receptor.
  • the native fiber protein of an adenoviral vector derived from serotype 5 adenovirus can be switched with the fiber protein from adenovirus serotypes 40 or 41.
  • the adenoviral vector can be modified to include an adenoviral coat protein (e.g., fiber, penton, or hexon protein) from a different serotype of adenovirus.
  • an adenoviral serotype 5 adenovirus can be modified to display an adenovirus serotype 35 fiber, which, in turn, can optionally comprise one or more non-native amino acid ligands . It is possible to utilize an adenoviral vector which does not naturally infect cell types of the inner ear to target the vector to a particular cell type.
  • an adenoviral vector which naturally transduces cells of the inner ear can be modified to display an adenoviral fiber protein and/or adenoviral penton base derived from an adenovirus which has no natural tropism for target cells, which adenoviral vector can display a non-native amino acid sequence that enables transduction of target cells.
  • the nucleic acid residues associated with native substrate binding can be mutated (see, e.g., WO 2000/15823; Einfeld, et al . (2001) J. Virol. 75 (23) : 11284-11291; van Beusechem, et al. (2002) J. Virol. 76 (6) : 2753-2762) such that the adenoviral vector incorporating the mutated nucleic acid residues is less able to bind its native substrate.
  • adenovirus serotypes 2 and 5 transduce cells via binding of the adenoviral fiber protein to the coxsackievirus and adenovirus receptor (CAR) and binding of penton proteins to integrins located on the cell surface.
  • the replication-deficient or conditionally-replicating adenoviral vector of the inventive method can lack native binding to CAR and/or exhibit reduced native binding to integrins.
  • the native CAR and/or integrin binding sites e.g., the RGD sequence located in the adenoviral penton base
  • adenoviral vector is selectively targeted to scarred epithelial cells (e.g., regions of the epithelium missing endogenous, functional hair cells) by ablation of native binding of the adenoviral vector to CAR and/or integrins and incorporation into the adenoviral capsid one or more non-native ligands.
  • scarred epithelial cells e.g., regions of the epithelium missing endogenous, functional hair cells
  • Suitable ligands that mediate transduction via a specific receptor can be determined using routine library display techniques (such as phage display) and include, for example, ligands bound by EGF and ligands from the FGF family of peptides.
  • Other examples of non-native amino acid sequences and their substrates include, but are not limited to, short (e.g., 6 amino acids or less) linear stretches of amino acids recognized by integrins, as well as polyamino acid sequences such as polylysine, polyarginine , etc.
  • Non- native amino acid sequences for generating chimeric adenoviral coat proteins are further described in US 6,455,314 and WO 2001/92549.
  • adenoviral vectors can be constructed and/or purified using the methods set forth, for example, in US 5,965,358, US 6,168,941, US 6,329,200, US 6,383,795, US 6,440,728, US 6,447,995, US 6,475,757, WO 1998/53087, WO 1998/56937, WO 1999/15686, WO 1999/54441, WO 2000/12765, WO 2001/77304, and WO 2002/29388, as well as the other references identified herein.
  • numerous expression vectors, including adenoviral vectors are available commercially.
  • Adeno-associated viral vectors can be constructed and/or purified using the methods set forth, for example, in US 4,797,368 and Laughlin, et al. (1983) Gene 23 : 65-73.
  • an expression vector for use in the inventive method depends on a variety of factors such as, for example, the host, immunogenicity of the vector, the desired duration of protein production, the target cell, and the like. As each type of expression vector has distinct properties, the inventive method can be tailored to any particular situation. Moreover, more than one type of expression vector can be used to deliver the nucleic acid sequence to the target cell.
  • the invention provides method of changing the sensory perception and preventing or treating hearing loss in an animal, wherein the method comprises administering to the inner ear at least two different expression vectors comprising a nucleic acid sequence encoding an atonal-associated factor and/or a nucleic acid sequence encoding an inhibitor of EGFR signaling.
  • the target cell in the inner ear e.g., a supporting cell
  • an adenoviral vector and an HSV vector in that adenoviral vectors efficiently transduce supporting cells and HSV vectors efficiently transduce neurons.
  • nucleic Acid Molecules harbors nucleic acid molecules, the expression of which facilitates the regeneration of hair cells and hearing restoration.
  • the nucleic acid molecules encode an atonal-associated factor and can further encode an inhibitor of EGFR signaling.
  • any transcription factor e.g., Mathl or Hathl or inhibitor of EGFR signaling
  • therapeutic fragments i.e., those fragments having biological activity sufficient to, for example, activate transcription
  • a fusion protein composed of a transcription factor or a therapeutic fragment thereof and, for example, a moiety that stabilizes peptide conformation, also can be present in the expression vector.
  • Nucleic acid molecules i.e., encoding an atonal- associated factor and/or an inhibitor of EGFR signaling
  • an expression cassette i.e., a particular base sequence that possesses functions which facilitate subcloning and recovery of a nucleic acid molecule (e.g., one or more restriction sites) or expression of a nucleic acid molecule (e.g., polyadenylation or splice sites) .
  • the nucleic acid molecule of interest e.g., encoding an atonal-associated factor and/or an inhibitor of EGFR signaling
  • the expression cassette is preferably inserted in a 3'->5' orientation, e.g., oriented such that the direction of transcription of the expression cassette is opposite that of the surrounding adenoviral genome.
  • the adenoviral vector can include multiple expression cassettes harboring nucleic acid molecules encoding the encoding atonal-associated factor and/or an inhibitor of EGFR signaling, wherein said cassettes can replace any of the deleted regions of the adenoviral genome.
  • the insertion of an expression cassette into the adenoviral genome can be facilitated by known methods, for example, by the introduction of a unique restriction site at a given position of the adenoviral genome.
  • the E3 region of the adenoviral vector is deleted, and the E4 region is replaced by a spacer element.
  • the nucleic acid molecule of interest is operably linked to regulatory sequences necessary for said expression, e.g., a promoter.
  • a "promoter” is a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis.
  • a nucleic acid molecule is "operably linked" to a promoter when the promoter is capable of directing transcription of that nucleic acid molecule.
  • a promoter can be native or non-native to the nucleic acid molecule to which it is operably linked. Any promoter (i.e., whether isolated from nature or produced by recombinant DNA or synthetic techniques) can be used in connection with the invention to provide for transcription of the nucleic acid molecule.
  • the promoter preferably is capable of directing transcription in a eukaryotic (desirably mammalian) cell.
  • the functioning of the promoter can be altered by the presence of one or more enhancers (e.g., the CMV immediate early enhancer) and/or silencers.
  • the invention preferentially employs a viral promoter.
  • Suitable viral promoters include, for instance, cytomegalovirus (CMV) promoters, such as the CMV immediate-early promoter, promoters derived from human immunodeficiency virus (HIV) , such as the HIV long terminal repeat promoter, Rous sarcoma virus (RSV) promoters, such as the RSV long terminal repeat, mouse mammary tumor virus (MMTV) promoters, HSV promoters, such as the Lap2 promoter or the herpes thymidine kinase promoter (Wagner, et al . (1981) Proc . Natl. Acad. Sci.
  • CMV cytomegalovirus
  • HMV Rous sarcoma virus
  • MMTV mouse mammary tumor virus
  • HSV promoters such as the Lap2 promoter or the herpes thymidine kinase promoter
  • promoters derived from SV40 or Epstein Barr virus promoters derived from SV40 or Epstein Barr virus, an adeno-associated viral promoter, such as the p5 promoter, and the like.
  • the viral promoter is an adenoviral promoter, such as the Ad2 or Ad5 major late promoter and tripartite leader, a CMV promoter (murine or human in origin), or an RSV promoter.
  • the promoter need not be a viral promoter.
  • the promoter can be a cellular promoter, i.e., a promoter that drives expression of a cellular protein.
  • Preferred cellular promoters for use in the invention will depend on the desired expression profile to produce the therapeutic agent (s).
  • the cellular promoter is preferably a constitutive promoter that works in a variety of cell types. Suitable constitutive promoters can drive expression of genes encoding transcription factors, housekeeping genes, or structural genes common to eukaryotic cells.
  • the Ying Yang 1 (YYl) transcription factor (also referred to as NMP-1, NF-El, and UCRBP) is a ubiquitous nuclear transcription factor that is an intrinsic component of the nuclear matrix (Guo, et al. (1995) Proc. Natl. Acad. Sci. USA 92:10526-10530) .
  • JEM-1 also known as HGMW and BLZF-1; Tong, et al. (1998) Leukemia 12 (11) : 1733-1740; Tong, et al . (2000) Genomics 69 (3) : 380-390
  • UbC ubiquitin promoter
  • promoters are constitutive promoters.
  • the promoter can be an inducible promoter, i.e., a promoter that is up- and/or down-regulated in response to appropriate signals.
  • suitable inducible promoter systems include, but are not limited to, the IL-8 promoter, the metallothionine inducible promoter system, the bacterial lacZYA expression system, the tetracycline expression system, and the T7 polymerase system.
  • promoters that are selectively activated at different developmental stages can be employed.
  • the promoter sequence that regulates expression of the nucleic acid molecule can contain at least one heterologous regulatory sequence responsive to regulation by an exogenous agent.
  • the regulatory sequences are preferably responsive to exogenous agents such as, but not limited to, drugs, hormones, or other gene products.
  • the regulatory sequences, e.g., promoter preferably are responsive to glucocorticoid receptor-hormone complexes, which, in turn, enhance the level of transcription of a therapeutic peptide or a therapeutic fragment thereof.
  • the promoter is a tissue-specific promoter, i.e., a promoter that is preferentially activated in a given tissue and results in expression of a gene product in the tissue where activated.
  • a tissue specific promoter for use in this invention can be chosen by the ordinarily skilled artisan based upon the target tissue or cell-type. Suitable promoters include, but are not limited to, BRN.3C, BRN 3.1, the POU ORF3 factor promoter, BRK1, BRK3, the chordin promoter, the noggin promoter, the jaggedl promoter, the jagged2 promoter, and the notchl promoter.
  • tissue-specific promoters for use in this invention are specific to supporting cells or sensory hair cells, such as an atonal promoter or a myosin Vila promoter, which function in hair cells, or a hes-1 promoter, which functions in supporting cells.
  • a promoter is selected that promotes transgene expression in scarred epithelium.
  • a promoter also can be selected for use in this invention by matching its particular pattern of activity with the desired pattern and level of expression of the desired protein (e.g., the atonal-associated factor).
  • a hybrid promoter can be constructed which combines the desirable aspects of multiple promoters.
  • a CMV-RSV hybrid promoter combining the CMV promoter's initial rush of activity with the RSV promoter's high maintenance level of activity is especially preferred for use in many embodiments of the inventive method. It is also possible to select a promoter with an expression profile that can be manipulated by an investigator.
  • the nucleic acid molecule further includes a polyadenylation site following the coding region of the nucleic acid molecule.
  • Any suitable polyadenylation sequence can be used, including a synthetic optimized sequence, as well as the polyadenylation sequence of BGH (Bovine Growth Hormone) , polyoma virus, TK (Thymidine Kinase) , EBV (Epstein Barr Virus) , and the papillomaviruses, including human papillomaviruses and BPV (Bovine Papilloma Virus) .
  • a preferred polyadenylation sequence is the SV40 (Human Sarcoma Virus-40) polyadenylation sequence. Also, preferably all the proper transcription signals (and translation signals, where appropriate) are correctly arranged such that the nucleic acid molecule is properly expressed in the cells into which it is introduced. If desired, the nucleic acid molecule also can incorporate splice sites (i.e., splice acceptor and splice donor sites) to facilitate mRNA production. Moreover, if the nucleic acid molecule encodes a protein or peptide, which is a processed or secreted protein or acts intracellularl , preferably the nucleic acid molecule further includes the appropriate sequences for processing, secretion, intracellular localization, and the like.
  • an especially, preferred method of modulating expression of a nucleic acid molecule involves the addition of site-specific recombination sites on the expression vector. Contacting an expression vector having site-specific recombination sites with a recombinase will either up- or down-regulate transcription of a coding sequence, or simultaneously up-regulate transcription of one coding sequence and down-regulate transcription of another, through the recombination event.
  • site- specific recombination to modulate transcription of a nucleic acid sequence is described in, for example, US 5,801,030, US 6,063,627 and WO 97/09439.
  • nucleic acid molecules encoding the atonal-associated factor and/or inhibitor of EGFR signaling can also encode an inhibitor of EGFR signaling.
  • the expression vector alternatively, or in addition, can include multiple expression cassettes encoding atonal-associated factor and/or an inhibitor of EGFR signaling.
  • the multiple coding sequences can be operably linked to different promoters, e.g., different promoters having dissimilar levels and patterns of activity. Alternatively, the multiple coding sequences can be operably linked to the same promoter to form a polycistronic element.
  • the invention also contemplates administering to the inner ear a cocktail of expression vectors, wherein each expression vectors encode an atonal-associated factor and/or an inhibitor of EGFR signaling.
  • the cocktail of expression vectors can further include different types of expression vectors, e.g., adenoviral vectors and adeno-associated viral vectors.
  • the invention further provides an adenoviral vector harboring a nucleic acid molecule (s) encoding an atonal-associated factor (e.g., Mathl or Hathl) and/or an inhibitor of EGFR signaling, wherein the nucleic acid molecule (s) is operably linked to regulatory sequences necessary for expression of the atonal-associated factor and/or inhibitor of EGFR signaling.
  • the adenoviral vector is deficient in at least one replication-essential gene function of at least the E4 region.
  • the nucleic acid molecule can be obtained from any source, e.g., isolated from nature, synthetically generated, isolated from a genetically engineered organism, and the like. Appropriate adenoviral vectors and regulatory sequences are discussed herein .
  • the invention further provides a method of generating a hair cell in differentiated sensory epithelia in vivo.
  • the method involves contacting differentiated sensory epithelial cells with an adenoviral vector (a) deficient in one or more replication-essential gene functions of the El region, the E4 region, and, optionally, one or more gene functions the E3 region, (b) having a spacer in the E4 region, and (c) harboring a nucleic acid molecule (s) encoding an atonal-associated factor and/or an inhibitor of EGFR signaling.
  • the nucleic acid molecule (s) is expressed to produce the atonal-associated factor and/or inhibitor of EGFR signaling such that a hair cell is generated.
  • adenoviral vector can be used to generate hair cells in vivo (and therefore is useful for prophylactically or therapeutically treat a hearing disorder or a balance disorder)
  • transdifferentiation of supporting cells can occur in vitro and, thus, can be used in methods of research.
  • a drug or expression vector of the inventive method ideally reaches the sensory epithelium of the inner ear.
  • the most direct routes of administration therefore, entail surgical procedures which allow access to the interior of the structures of the inner ear.
  • Inoculation via cochleostomy allows administration of an expression vector directly to the regions of the inner ear associated with hearing.
  • Cochleostomy involves drilling a hole through the cochlear wall, e.g., in the otic capsule below the stapedial artery as described in Kawamoto, et al. ((2001) Molecular Therapy 4 ( 6) : 575-585 ) , and release of a pharmaceutical composition containing the drug or expression vector.
  • Administration to the endolymphatic compartment is particularly useful for administering an adenoviral vector to the areas of the inner ear responsible for hearing.
  • a drug or expression vector can be administered to the semicircular canals via canalostomy. Canalostomy provides for transgene expression in the vestibular system and the cochlea, whereas cochleostomy does not provide as efficient transduction in the vestibular space. The risk of damage to cochlear function is reduced using canalostomy in as much as direct injection into the cochlear space can result in mechanical damage to hair cells (Kawamoto, et al . , supra) .
  • Administration procedures also can be performed under fluid (e.g., artificial perilymph), which can include factors to alleviate side effects of treatment or the administration procedure, such as apoptosis inhibitors or anti ⁇ inflammatories .
  • Another direct route of administration to the inner ear is through the round window, either by injection or topical application to the round window.
  • Administration via the round window is especially preferred for delivering a drug or adenoviral vector to the perilymphatic space.
  • Transgene expression in cochlear and vestibular neurons and cochlear sensory epithelia has been observed following administration of expression vectors via the round window
  • an adenoviral vector can display one or more ligands that enhance uptake of the adenoviral vector into target cells
  • the adenoviral vector can encode one or more adenoviral coat proteins which are modified to reduce native binding (e.g., CAR- and/or integrin-binding) and harbor a non-native amino acid sequence which enhances uptake of the adenoviral vector by target cells of the inner ear.
  • native binding e.g., CAR- and/or integrin-binding
  • a drug or expression vector ⁇ e.g., adenoviral vector can be present in a pharmaceutical composition for administration to the inner ear.
  • a drug or expression vector can be present in or on a device that allows controlled or sustained release of the drug or expression vector, such as a sponge, meshwork, mechanical reservoir or pump, or mechanical implant.
  • a biocompatible sponge or gelform soaked in a pharmaceutical composition containing the drug or expression vector is placed adjacent to the round window, through which the drug or expression vector permeates to reach the cochlea (as described in Jero, et al., supra) .
  • Mini-osmotic pumps provide sustained release of a drug or expression vector over extended periods of time (e.g., five to seven days) , allowing small volumes of composition containing the drug or expression vector to be administered, which can prevent mechanical damage to endogenous sensory cells.
  • the drug or expression vector also can be administered in the form of sustained-release formulations (see, e.g., US 5,378,475) containing, for example, gelatin, chondroitin sulfate, a polyphosphoester, such as bis-2-hydroxyethyl-terephthalate (BHET) , or a polylactic-glycolic acid.
  • sustained-release formulations see, e.g., US 5,378,475
  • the drug or expression vector can be administered parenterally, intramuscularly, intravenously, orally or intraperitoneally .
  • a drug or expression vector that is parenterally administered to a patient for generating sensory hair cells in the ear is specifically targeted to sensory epithelial cells, such as supporting cells.
  • the expression vector is targeted to scarred sensory epithelium to promote generation of exogenous hair cells to replace damaged endogenous hair cells.
  • an expression vector can be modified to alter the binding specificity or recognition of an expression vector for a receptor on a potential host cell. With respect to adenovirus, such manipulations can include deletion of regions of the fiber, penton, or hexon, insertions of various native or non- native ligands into portions of the coat protein, and the like.
  • parenteral administration can require large doses or multiple administrations to effectively deliver the expression vector to the appropriate host cells.
  • compositions are well-known to those of ordinary skill in the art (see Pharmaceutics and Pharmacy Practice, (1982) J.B. Lippincott Co., Philadelphia, PA, Banker and Chalmers, eds . , pages 238-250; ASHP Handbook on Injectable Drugs (1986) Toissel, 4 th ed., pages 622-630).
  • the expression vector can also be administered in vivo by particle bombardment, i.e., a gene gun.
  • null expression vectors i.e., an expression vector not harboring the nucleic acid molecule (s) of interest
  • Prior administration of null expression vectors can serve to create an immunity in the host to the expression vector hinder the body's innate clearance mechanisms, thereby decreasing the amount of vector cleared by the immune system.
  • Dosage The dose of a drug or expression vector administered to an animal, particularly a human, in accordance with the invention should be sufficient to affect the desired response in the animal over a reasonable time frame.
  • dosage will depend upon a variety of factors, including the age, species, location of damaged sensory epithelia, the pathology in question (if any) , and condition or disease state. Dosage also depends on the atonal-associated factor, inhibitor of EGFR signaling and/or cell cycle-associated protein kinase inhibitor, as well as the amount of sensory epithelium to be transduced.
  • the size of the dose also will be determined by the route, timing, and frequency of administration as well as the existence, nature, and extent of any adverse side effects that might accompany the administration of a particular expression vector (e.g., surgical trauma) or drug and the desired physiological effect. It will be appreciated by one of ordinary skill in the art that various conditions or disease states, in particular, chronic conditions or disease states, may require prolonged treatment involving multiple administrations .
  • Suitable doses and dosage regimens can be determined by conventional range-finding techniques known to those of ordinary skill in the art.
  • the expression vector is a viral vector, most preferably an adenoviral vector
  • about 10 5 viral particles to about 10 12 viral particles are delivered to the patient.
  • a pharmaceutical composition can be administered that includes an expression vector concentration of about 10 5 particles/ml to about 10 13 particles/ml (including all integers within the range of about 10 s particles/ml to about 10 13 particles/ml) , preferably about 10 10 particles/ml to about 10 12 particles/ml, and will typically involve the administration of about 0.1 ⁇ to about 100 ⁇ of such a pharmaceutical composition directly to the inner ear.
  • the dose of one administration preferably is at least about lxlO 6 particles (e.g., about 4xl0 6 -4*10 12 particles), more preferably at least about 1*10 7 particles, more preferably at least about 1*10 8 particles (e.g., about 4xl0 8 -4xl0 1:L particles), and most preferably at least about lxlO 9 particles to at least about lxlO 10 particles (e.g., about 4xl0 9 -4xl0 10 particles) of an adenoviral vector harboring a nucleic acid molecule encoding an atonal- associated factor and/or a co-transcription factor and/or inhibitor of a gene silencing complex.
  • lxlO 6 particles e.g., about 4xl0 6 -4*10 12 particles
  • 1*10 7 particles more preferably at least about 1*10 8 particles (e.g., about 4xl0 8 -4xl0 1:L particles)
  • the dose of the pharmaceutical composition includes no more than about lxlO 14 particles, preferably no more than about lxlO 13 particles, even more preferably no more than about lxlO 12 particles, even more preferably no more than about lxlO 11 particles, and most preferably no more than about lxlO 10 particles (e.g., no more than about lxlO 9 particles) .
  • a single dose of pharmaceutical composition can be about lxlO 6 particle units (pu) , 4 ⁇ 10 6 pu, lxlO 7 pu, 4xl0 7 pu, lxlO 8 pu, 4xl0 8 pu, lxlO 9 pu, 4 ⁇ 10 9 pu, lxlO 10 pu, 4xl0 10 pu, lxlO 11 pu, 4xlO pu, lxlO 11 pu, 4xlO pu, lxlO 12 pu, or 4xl0 12 pu of the adenoviral vector (e.g., the replication-deficient adenoviral vector) .
  • the adenoviral vector e.g., the replication-deficient adenoviral vector
  • the expression vector is a plasmid
  • preferably about 0.5 ng to about 1000 ⁇ g of DNA is administered. More preferably, about 0.1 ⁇ g to about 500 is administered, even more preferably about 1 pg to about 100 ⁇ g of DNA is administered. Most preferably, about 50 pg of DNA is administered to the inner ear.
  • other routes of administration may require smaller or larger doses to achieve a therapeutic effect. Any necessary variations in dosages and routes of administration can be determined by the ordinarily skilled artisan using routine techniques known in the art .
  • the interior space of the structures of the inner ear is limited.
  • the volume of pharmaceutical composition administered directly into the inner ear structures should be carefully monitored, as forcing too much composition will damage the sensory epithelium.
  • the volume administered is preferably about 10 ⁇ to about 2 ml (e.g., from about 25 ⁇ to about 1.5 ml) of composition.
  • from about 50 ⁇ to about 1 ml (e.g., about 100 ⁇ , 200 ⁇ , 300 ⁇ , 400 ⁇ , 500 ⁇ , 600 ⁇ , 700 ⁇ , 800 ⁇ or 900 ⁇ ) of composition can be administered.
  • the entire fluid contents of the inner ear structure e.g., the cochlea or semicircular canals, is replaced with pharmaceutical composition.
  • a pharmaceutical composition of the invention is slowly released into the inner ear structure, such that mechanical trauma is minimized .
  • the inventive method provides for administration of multiple doses of a drug or expression vector to generate hair cells in the sensory epithelium to change the sensory perception of an animal.
  • at least two doses of a drug or expression vector can be administered to the same ear.
  • the multiple doses are administered while retaining gene expression above background levels.
  • the sensory epithelium of the inner ear is contacted with two doses or more of the drug or expression vector within about 30 days.
  • two or more applications are administered to the inner ear within about 90 days.
  • three, four, five, six, or more doses can be administered in any time frame (e.g., 2, 7, 10, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 85 or more days between doses) .
  • a drug or expression vector of the invention desirably is administered in a pharmaceutical composition, which includes a pharmaceutically acceptable carrier and the drug or expression vector (s).
  • a pharmaceutically acceptable carrier can be used within the context of the invention, and such carriers are well known in the art.
  • the choice of carrier will be determined, in part, by the particular site to which the composition is to be administered and the particular method used to administer the composition.
  • the pharmaceutical composition preferably is free of replication-competent adenovirus.
  • Suitable formulations include aqueous and non ⁇ aqueous solutions, isotonic sterile solutions, which can contain anti-oxidants , buffers, bacteriostats , and solutes that render the formulation isotonic with the blood or fluid of the inner ear of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers , thickening agents, stabilizers, and preservatives.
  • the formulation can include artificial endolymph or perilymph, which are commercially available.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried ( lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, immediately prior to use.
  • sterile liquid carrier for example, water
  • Extemporaneous solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • the pharmaceutically acceptable carrier is a buffered saline solution.
  • the expression vector for use in the inventive method is administered in a pharmaceutical composition formulated to protect the expression vector from damage prior to administration.
  • the pharmaceutical composition can be formulated to reduce loss of the expression vector on devices used to prepare, store, or administer the expression vector, such as glassware, syringes, or needles.
  • the pharmaceutical composition can be formulated to decrease the light sensitivity and/or temperature sensitivity of the expression vector.
  • the pharmaceutical composition preferably includes a pharmaceutically acceptable liquid carrier, such as, for example, those described above, and a stabilizing agent selected from the group consisting of polysorbate 80, L- arginine, polyvinylpyrrolidone, trehalose, and combinations thereof.
  • a pharmaceutical composition also can be formulated to enhance transduction efficiency.
  • the expression vector e.g., viral vector
  • the expression vector can be present in a composition with other therapeutic or biologically-active agents.
  • therapeutic factors useful in the treatment of a particular indication can be present.
  • Factors that control inflammation such as ibuprofen or steroids, can be part of the composition to reduce swelling and inflammation associated with in vivo administration of the viral vector.
  • Immune system suppressors can be administered in combination with the inventive method to reduce any immune response to the vector itself or associated with a disorder of the inner ear.
  • Angiogenic factors, neurotrophic factors, proliferating agents, and the like can be present in the pharmaceutical composition.
  • vitamins and minerals, anti-oxidants , and micronutrients can be co ⁇ administered.
  • Antibiotics i.e., microbicides and fungicides, can be present to reduce the risk of infection associated with gene transfer procedures and other disorders .
  • the inventive method includes administering an inhibitor of EGFR signaling and/or an expression vector (s) harboring a nucleic acid molecule (s) encoding an atonal-associated factor to change the sensory perception of an animal by generating hair cells in the sensory epithelium of the inner ear.
  • the nucleic acid molecule encoding the atonal-associated factor can encode multiple (i.e., two, three, or more) atonal-associated factors and/or inhibitors of EGFR signaling.
  • the mere generation of a hair cell does not ensure a change in sensory perception in an animal.
  • a sufficient number of hair cells should be generated, and those sensory hair cells should be linked to a neural network capable of transmitting signals to the brain. Accordingly, while not required, it may be advantageous to provide additional factors to ensure proper reception and transmission of signals to the brain.
  • the nucleic acid molecule (s) encoding the atonal-associated factor and/or inhibitor of EGFR signaling can encode additional gene products.
  • the expression vector alternatively, or in addition, can include inulLiple expression cassettes encoding different gene products.
  • Multiple coding sequences can be operably linked to different promoters, e.g., different promoters having dissimilar levels and patterns of activity. Alternatively, the multiple coding sequences can be operably linked to the same promoter to form a polycistronic element.
  • the invention also contemplates administering to the inner ear a cocktail of expression vectors, wherein each expression vector encodes an atonal-associated factor or another gene product beneficial to sensory perception.
  • the cocktail of expression vectors can further comprise different types of expression vectors, e.g., adenoviral vectors and adeno- associated viral vectors.
  • the inventive method also includes the administration of an expression vector harboring a nucleic acid molecule encoding an atonal-associated factor in combination with an inhibitor of EGFR signaling, which is not encoded by an expression vector.
  • the method and pharmaceutical compositions disclosed herein can be modified to include the expression vector harboring a nucleic acid molecule encoding an atonal-associated factor in combination with an isolated inhibitory RNA molecule, protein, peptide, antibody, or small organic molecule that inhibits EGFR signaling.
  • compositions disclosed herein can be modified to include (i) the expression vector (s) harboring a nucleic acid molecule (s) encoding an atonal-associated factor and/or an inhibitor of EGFR signaling in combination with (ii) an isolated inhibitory RNA molecule, protein, peptide, antibody, or small organic molecule that inhibits EGFR signaling.
  • the kit includes a nucleic acid molecule encoding an atonal-associated factor, and (i) a nucleic acid molecule encoding an inhibitor of epidermal growth factor receptor (EGFR) signaling; (ii) an isolated inhibitor of EGFR signaling (e.g., an inhibitory RNA or small organic molecule that inhibits one or more of EGFR, Ras, Raf, MEK, ERK/MAPK, JAK, STAT, PI3K, AKT, mTOR, NCK, PAK, JNK, PLC, PKC or a cell cycle associated kinase) ; or (iii) a combination of (i) and (ii) .
  • EGFR epidermal growth factor receptor
  • an isolated inhibitor of EGFR signaling e.g., an inhibitory RNA or small organic molecule that inhibits one or more of EGFR, Ras, Raf, MEK, ERK/MAPK, JAK, STAT, PI3K, AKT, mTOR
  • the kit can include containers (e.g., vials, bottles, syringes, or tubes) containing the active ingredients in lyophilized or liquid form as well as instructions for using the kit components including information regarding dosing, administration, timing of administration and the like.
  • the kit may further include an indication of the active ingredients, reference to scientific literature, packing materials, clinical trial results, and the like. The information may be based on the results of various studies, for example, studies using experimental animals involving in vivo models and studies based on human clinical trials.
  • the inventive method also contemplates delivery of a nucleic acid molecule encoding at least one neurotrophic agent.
  • the neurotrophic agent is a neural growth stimulator, which induces growth, development, and/or maturation of neural processes.
  • Neurotrophic factors also can be administered to protect or maintain existing and developing neurons. For a newly generated hair cell to function properly, a neural network should be in place to transmit neural impulses to the brain. Accordingly, it is advantageous to protect existing neurons associated with the sensory epithelium of the inner ear while generating new hair cells, induce the growth and maturation of new neural processes, and/or simply direct existing neural processes to sensory hair cells .
  • Neurotrophic factors are divided into three subclasses: neuropoietic cytokines; neurotrophins ; and the fibroblast growth factors.
  • Ciliary neurotrophic factor (CNTF) is exemplary of neuropoietic cytokines. CNTF promotes the survival of ciliary ganglionic neurons and supports certain neurons that are NGF-responsive .
  • Neurotrophins include, for example, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) , which stimulates neurite outgrowth.
  • Other neurotrophic factors include, for example, transforming growth factors, glial cell-line derived neurotrophic factor (GDNF) , neurotrophin 3, neurotrophin 4/5, and interleukin 1- ⁇ .
  • Neuronotrophic factors enhance neuronal survival and also are suitable for use in the inventive method. It has been postulated that neuronotrophic factors can actually reverse degradation of neurons. Such factors, conceivably, are useful in treating the degeneration of neurons associated with age, infection, or trauma.
  • a preferred neuronotrophic factor is pigment epithelium derived factor (PEDF) . PEDF is further described in Chader (1987) Cell Different. 20:209- 216; Pignolo, et al. (1998) J. Biol. Chem. 268 ( 12 ): 8949- 8957; US 5,840,686, WO 1993/24529, WO 1999/04806, and WO 2001/58494.
  • Proliferating agents induce cellular proliferation, preferably proliferation of supporting cells in the inner ear. Multiplying the number of hair cell progenitors maximizes the biological effect of the atonal-associated factor and/or a co-transcription factor and/or inhibitor of a gene silencing complex. Supporting cell proliferation is induced by mitogenic growth factors, such as fibroblast growth factors (FGF, in particular FGF-2), vascular endothelial growth factors (VEGF) , epidermal growth factor (EGF) , E2F, cell cycle up-regulators , and the like.
  • FGF fibroblast growth factors
  • VEGF vascular endothelial growth factors
  • EGF epidermal growth factor
  • E2F cell cycle up-regulators
  • a nucleic acid sequence encoding a proliferating agent can be administered in conjunction with the nucleic acid molecule (s) encoding an atonal-associated factor and/or an inhibitor of EGFR signaling in the inventive method.
  • the nucleic acid molecule encoding a proliferating agent can be engineered to exert its biological effect only on the cell type to be replicated.
  • the nucleic acid can include a regulatory sequence that is preferentially activated in supporting cells.
  • the resulting proliferating agent also can be engineered to prevent secretion into the cellular milieu.
  • a substance can be administered to the inner ear to promote cell proliferation or enhance uptake of the expression vector .
  • the method of the invention can be part of a treatment regimen involving other therapeutic modalities. It is appropriate, therefore, if the inventive method is employed to prophylactically or therapeutically treat a sensory disorder, namely a hearing disorder or a balance disorder, that has been treated, is being treated, or will be treated with any of a number of other therapies, such as drug therapy or surgery.
  • the inventive method also can be performed in conjunction with the implantation of hearing devices, such as cochlear implants.
  • the inventive method also is particularly suited for procedures involving stem cells to regenerate populations of cells within the inner ear. In this respect, the inventive method can be practiced ex vivo to transduce stem cells, which are then implanted within the inner ear.
  • the expression vector is preferably administered as soon as possible after it has been determined that an animal, such as a mammal, specifically a human, is at risk for degeneration of sensory hair cells (prophylactic treatment) or has demonstrated reduced numbers or damage of sensory hair cells (therapeutic treatment) .
  • Treatment will depend, in part, upon the particular nucleic acid molecule used, the particular atonal-associated factor and/or inhibitor of EGFR signaling expressed, the expression vector, the route of administration, and the cause and extent, if any, of hair cell loss or damage realized.
  • An expression vector (s) harboring a nucleic acid molecule (s) encoding an atonal-associated factor and/or an inhibitor of EGFR signaling can be introduced ex vivo into cells previously removed from a given animal, in particular a human.
  • Such transduced autologous or homologous host cells can be progenitor cells that are reintroduced into the inner ear of the animal or human to express the atonal- associated factor and/or inhibitor of EGFR signaling and differentiate into mature hair cells in vivo.
  • progenitor cells that are reintroduced into the inner ear of the animal or human to express the atonal- associated factor and/or inhibitor of EGFR signaling and differentiate into mature hair cells in vivo.
  • One of ordinary skill in the art will understand that such cells need not be isolated from the patient, but can instead be isolated from another individual and implanted into the patient .
  • the inventive method also can involve the co ⁇ administration of other pharmaceutically active compounds.
  • co-administration is meant administration before, concurrently with, e.g., in combination with the expression vector in the same formulation or in separate formulations, or after administration of the expression vector as described above.
  • factors that control inflammation such as ibuprofen or steroids, can be co ⁇ administered to reduce swelling and inflammation associated with administration of the expression vector.
  • Immunosuppressive agents can be co-administered to reduce inappropriate immune responses related to an inner ear disorder or the practice of the inventive method.
  • vitamins and minerals, anti-oxidants , and micronutrients can be co-administered.
  • Antibiotics i.e., microbicides and fungicides, can be co-administered to reduce the risk of infection associated with surgical procedures .
  • Example 1 Small Molecule Inhibition of EGFR Signaling for Hair Cell Regeneration
  • Atohl a transcription factor, can convert non- sensory supporting cells (SCs) to hair cells (HCs) in neonatal and juvenile mammalian cochleae (Izumikawa, et al . (2005) Nature Med. 11:271-6; Kelly, et al. (2012) J. Neurosci. 32:6699-710; Liu, et al . (2012) J. Neurosci. 32:6600-10).
  • SCs non- sensory supporting cells
  • HCs hair cells
  • Ectopic expression of Atohl has been approved for clinical trials as a gene therapy.
  • Atohl- induced HC conversion is inefficient ( ⁇ 17%), incomplete (lacking mature HC markers), and age-dependent (no response in adult cochlea) (Izumikawa, et al .
  • RNA-seq analysis to compare the transcriptome of SCs, endogenous outer HCs (OHCs), and Atohl-induced HCs (cHCs) in adult mouse cochleae, it was observed that the epidermal growth factor receptor (EGFR) signaling is enriched in SCs and cHCs using gene set enrichment analysis (GSEA) .
  • GSEA gene set enrichment analysis
  • Proxl-CreER Proxl
  • CAG-loxp-stop-loxp-Atohl-HA HA
  • EGFR flox/flox were crossed to generate an inducible mouse line ( Proxl ; HA; EGFR flo /flox or PHER) in which Atohl overexpression together with EGFR deletion can be achieved specifically in neonatal and juvenile SCs (Deiters cells (DC) and pillar cells (PC) underneath outer HCs (OHCs)) upon tamoxifen induction.
  • DC Deiters cells
  • PC pillar cells
  • OHCs outer HCs
  • Glast (Glast; HA; EGFR flox/flox or GHER) that specifically targets inner phalangeal and inner border cells underneath inner HCs (IHCs) .
  • Fgfr3-CreER; HA; EGFR flox flox or FHER mice were generated to target adult deiter and pillar cells. Cre activity will be induced at P28 adult age and HC regeneration 3 and 6 weeks later will be investigated. It is expected that an increase in SC conversion to HC will be observed in mice deficient in EGFR expression.
  • mice will be exposed to 8-16 kHz octave band noise at 120-dB SPL for 2 hours to induce OHC loss and hearing loss. Subsequently, HC regeneration will be induced with tamoxifen 7 days post ⁇ exposure. The ABR of the animals will be tested before noise damage, 7 days after noise damage, and 3 weeks after tamoxifen induction to further determine molecular pathways in HC regeneration in the mouse models .
  • Selected EGFR inhibitors will be tested in mouse models with Atohl overexpression (PH, GH, and FH) to ascertain their potential to mimic the genetic models described above in Example 2 (i.e., PHER, GHER, and FHER respectively) .
  • Six highly potent and specific EGFR inhibitors will be individually tested including Erlotinib, Gefitinib, Afatinib, NT-113, AZD3759, and Dacomitinib, with EC 5 o values of 0.2-33 nM. These compounds have been approved by the FDA or are in clinical trials for other diseases, and have relatively well-characterized absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties across species.
  • ADMET absorption, distribution, metabolism, excretion, and toxicity
  • Atohl overexpression will be induced in SCs with tamoxifen in the mouse models (PH, GH, and FH) . Subsequently, the selected compounds will be delivered by IP or TT injection. ADMET properties of each compound in plasma, cochlear homogenate, or perilymph will be assessed, and exposure-efficacy relationships in regenerating functional HCs and restoring hearing after NIHL will be examined.
  • EGFRi/Atohl EGFR inhibitors together with overexpression of Atohl
  • EGFRi/Atohl EGFR inhibitors together with overexpression of Atohl
  • a portion of these HCs will express mature HC markers (prestin, oncomodulin, vGlut3) and exhibit similar electrophysiology as normal mature HCs.
  • EGFRi/Atohl-mediated functional HC conversion occurs even in noise-induced hearing loss.
  • an Atohl/Pou4f3 overexpressing mouse model can be used, which gives rise to -150 cHCs (highest among many models) per cochlea from SCs when induced at adult ages. It is expected that the EGFR inhibition will synergistically promote SC- to-HC conversion with overexpression of both Pou4f3 and Atohl .
  • Example 4 EGFR Inhibitors for Protection Against Hearing Loss
  • a screen of a library composed of 75 kinase inhibitors was conducted to identify inhibitors that protect against cisplatin-induced hair cell loss.
  • This screen identified four compounds: (1) Her2 inhibitor UBRITINIB (TAK 165), (2) Pan-AUR inhibitor SNS314 (3) BRAF-V600E inhibitor GSK2118436A (DABRAFENIB) , and (4) PDGFR inhibitor CRENOLANIB that potently protected against cisplatin-induced cell death in a mouse cochlea-derived cell line (HEI-OC1) as well as cisplatin-induced hair cell loss in cochlear explants.
  • Her2 inhibitor UBRITINIB TAK 165
  • Pan-AUR inhibitor SNS314 (3) BRAF-V600E inhibitor GSK2118436A (DABRAFENIB)
  • PDGFR inhibitor CRENOLANIB that potently protected against cisplatin-induced cell death in a mouse cochlea-derived cell line (HEI-OC1) as well as
  • Her2 inhibitor MUBRITINIB (TAK 165) exhibited protective effects against cisplatin-induced Caspase-3/7 activity in HEI-OC1 cells with an IC 50 of 4 nM and LD 50 of >55 ⁇ ; and protected against cisplatin-induced hair cell loss in mouse cochlear explants with IC 50 of 2.5 nM and LD 50 of >500 nM (Therapeutic Index of >200) ( Figure 3) .
  • the pan-ErbB inhibitor, PELITINIB was found to exhibit protective effects against cisplatin-induced Caspase-3/7 activity in HEI-OCl cell loss with IC 50 of 0.6 ⁇ and LD 50 of 40 ⁇ ( Figure 4) .
  • BLB blood-labyrinth barrier
  • Oral and other routes may also be considered for in vivo properties ⁇ e.g., solubility, permeability, pharmacokinetics/pharmacodynamics (PK/PD), and absorption, distribution, metabolism, excretion, and toxicology (ADMET) ) .
  • Compounds to test can be selected on the basis of: (1) exhibiting potent IC 50 values and minimal toxicity (i.e., high LD 5 o/IC 5 o values, preferably >50-100 ⁇ ) ; (2) targeting several different biological targets /pathways ; and (3) capacity to be delivered via other routes (e.g., oral) .
  • mice exposed to noise or blast exposure are immediately treated with the individual compounds in one ear and vehicle control (0.5% DMSO) in the other ear.
  • DMSO or compound are delivered locally at the highest feasible dose (which should be much higher than the IC 50 in cochlear explants but not toxic by itself in vivo) by trans-tympanic injection into the adult mouse middle ear at ⁇ 5 pL per ear.
  • the ABR and Distortion Products Otoacoustic Emissions (DPOAE) are measured pre-noise exposure or TBI and at 1 and 2 weeks post-injection. After hearing tests by ABR and DPOAE, the mice are cardiac- perfused for fixation and harvesting of the cochleae.
  • the cochleae are analyzed using both whole-mount preparations and sections, and immunofluorescence is used to detect HC/SC markers (i.e., phalloidin, Myo7a, Prestin, and Sox2, etc.) and synaptic markers (Ctbp2, GluR2/3 and Tujl) (Liu, et al. (2014) PLoS One 9:e89377).
  • HC/SC markers i.e., phalloidin, Myo7a, Prestin, and Sox2, etc.
  • synaptic markers Ctbp2, GluR2/3 and Tujl
  • the entire procedure is double-blinded: one person encodes DMSO or compound while the other person 1 randomly injects the left and right ears of the same mouse, and the person who records ABR and DPOAE does not know which ear was injected with compound until the entire experiment is completed .
  • ABR measurements have been previously described in detail (Dallos, et al. (2008) Neuron 58:333-339; Gao, et al. (2007) Mol. Cell Biol. 27:4500-4512; Liberman, et al . (2002) Nature 419:300-304; Liu, et al . (2014) PLoS One 9:e89377; Wu, et al . (2004) Brain Res. Mol. Brain Res. 126:30-37; Yamashita, et al . (2012) PLoS One 7:e45453) .
  • mice are anesthetized by intraperitoneal injection of AVERTIN (0.5 mg/kg body weight) and placed on an electric heating pad to maintain body temperature, using a homeothermic blanket system (Harvard Apparatus Ltd) . Mice that die or show signs of middle-ear dysfunction during the course of the experiment are excluded from analysis. All recordings are conducted in a sound booth (Industrial Acoustic Company) . For acoustic stimulation and measurements, two speakers (fl and f2; ECl) and a microphone (ER-10B, Etymotic Research, Elk Grove Village, IL) are connected to a short flexible coupler tube with a tapered plastic tip that is inserted into the external auditory meatus.
  • AVERTIN 0.5 mg/kg body weight
  • ECl ECl
  • ER-10B Etymotic Research, Elk Grove Village, IL
  • the microphone is calibrated in situ with the coupler in the measuring position. At frequencies higher than 22 kHz, the frequency responses of the measurement microphone (ER10B+) are lower than those of a reference microphone (ACO-7017; ACO Pacific, Inc., Belmont, CA) . Therefore, DPOAE 2fl-f2 responses are recorded at a frequency fl range of 5454-18180 Hz, using the TDT BioSig III system (TDT) . Signal duration is 83.88 ms, with a repetition rate of 11.92/s. The fl and f2 responses are passed separately through an RX6 MultiFunction Processor (TDT) for digital/analog conversion to PA5 programmable attenuators.
  • TDT TDT BioSig III system
  • Stimulus intensity is reduced from 90 to 0 dB in 5 dB steps to establish thresholds and is digitally sampled at 200 kHz and averaged from 100 discrete spectra.
  • the signals are delivered through EDI speaker drivers that feed into the ECl electrostatic speakers coupled to the ear canal.
  • the resulting ear canal sound pressure is recorded with an ER10B+ low noise microphone (gain O x ) and probe (Etymotic) housed in the same coupler as the fl and f2 speakers.
  • the output of the ER10B+ amplifier is routed directly to an RX6 MultiFunction Processor (TDT) for analog/digital conversion for sampling at 200 kHz.
  • TTT RX6 MultiFunction Processor
  • FFT Fast- Fourier transforms
  • mice are anesthetized by intraperitoneal (i.p.) injection of AVERTIN or ketamine and xylazine. Body temperature is maintained on a heating pad during the surgical procedure. Lubricant eye ointment is applied to prevent corneal ulcers, as the blinking reflex disappears during surgery. The tympanic membrane is visualized with a surgical stereomicroscope. Using a 33-gauge cannula, 5 pL of compound or DMSO in PBS is gently injected through the tympanic membrane, followed by surgical stereomicroscopic confirmation that the solution is in the middle ear cavity. Mice are then placed in the cage on the heating pad for an additional 30 minutes . After surgery, all mice are allowed to recover on a heating pad before being returned to the animal housing facility .

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Abstract

L'invention concerne des procédés et des compositions utilisant un inhibiteur de la signalisation EGFR pour la prévention de la perte auditive ou un inhibiteur de la signalisation EGFR et une molécule d'acide nucléique codant pour un facteur lié à l'atonal pour le traitement de la perte auditive.
PCT/US2018/030114 2017-05-03 2018-04-30 Compositions et procédés de prévention et de traitement de perte d'audition WO2018204226A1 (fr)

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CN201880029618.7A CN111655228B (zh) 2017-05-03 2018-04-30 预防和治疗听力损失的组合物和方法
JP2019560674A JP7291399B2 (ja) 2017-05-03 2018-04-30 難聴の予防および治療のための組成物および方法
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Publication number Priority date Publication date Assignee Title
US11013752B2 (en) 2016-06-03 2021-05-25 Hough Ear Institute Combination therapies for inner ear sensory hair cell regeneration/replacement
WO2021118924A2 (fr) 2019-12-12 2021-06-17 Ting Therapeutics Llc Compositions et méthodes de prévention et de traitement de la perte d'audition
WO2021118924A3 (fr) * 2019-12-12 2021-08-05 Ting Therapeutics Llc Compositions et méthodes de prévention et de traitement de la perte d'audition
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CN114981298B (zh) * 2019-12-12 2024-08-20 听治疗有限责任公司 用于预防和治疗听力损失的组合物和方法
WO2021163610A1 (fr) * 2020-02-13 2021-08-19 Spiral Therapeutics Inc. Traitement de maladies et d'affections otiques
US11857551B1 (en) 2020-07-10 2024-01-02 Ting Therapeutics Llc Methods for the prevention and treatment of hearing loss
CN114762733A (zh) * 2021-01-15 2022-07-19 中国科学院脑科学与智能技术卓越创新中心 耳蜗外毛细胞特异性顺式调节元件及其应用

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